Diagnostic assays and kits for body mass and cardiovascular disorders

ABSTRACT

The present invention is based at least in part on the discovery of the genomic structure of the human SR-BI gene and on the identification of polymorphic regions within the gene. Accordingly, the invention provides nucleic acids having a nucleotide sequence of an allelic variant of an SR-BI gene and nucleic acids having an SR-BI intronic sequence. The invention also provides methods for identifying specific alleles of polymorphic regions of an SR-BI gene, methods for determining whether a subject has or is at risk of developing a disease which is associated with a specific allele of a polymorphic region of an SR-BI gene, and kits for performing such methods.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 09/031,626, filed on Feb. 27, 1998, which is acontinuation-in-part of U.S. Application No. 08/890,979, filed on Jul.10, 1997 (U.S. Pat. No. 6,030,778), the contents of which areincorporated herein in their entirety by this reference.

BACKGROUND OF THE INVENTION

[0002] Coronary heart disease is a major health risk throughout theindustrialized world. Atherosclerosis, the most prevalent ofcardiovascular diseases, is the principal cause of heart attack, stroke,and gangrene of the extremities, and thereby the principle cause ofdeath in the United States. Dyslipidemia is associated with thedevelopment of coronary heart disease (CHD) and atherosclerosis.Although historically much emphasis has been placed on total plasmacholesterol levels as a risk factor for coronary heart disease, it hasbeen clearly established that low levels of high density lipoproteincholesterol (HDL) is an independent risk factor for this disease. Familyand twin studies have shown that there are genetic components thataffect HDL levels. However, mutations in the main protein components ofHDL (ApoA1 and ApoAII) and in the enzymes that are known to be involvedin HDL metabolism (e.g., CETP, HL, LPL and LCAT) do not explain all ofthe genetic factors affecting HDL levels in the general population (J.L. Breslow, in The Metabolic and Molecular Bases of Inherited Disease,C. R. Scriver, A. L. Beaudet, W. Sly, D. Valle, Eds. (McGraw-Hill, NewYork, 1995), pp 2031-2052; and S. M. Grundy, (1995) J Am. Med. Assoc.256: 2849). This finding in combination with the fact that themechanisms of HDL metabolism are poorly understood, suggests that thereare other as yet unknown factors that contribute to the geneticvariability of lipid levels, including HDL levels.

[0003] Another disorder that is often associated with high triglycerideand low high density lipoprotein (HDL) concentrations is obesity, whichrenders a subject susceptible to cardiovascular diseases, such asischemia, restenosis, congestive heart failure, and atherosclerosis.Severely obese individuals (weighing 60% over a normal weight) have ahigh risk of developing cardiorespiratory problems. They are also atrisk of developing chronic hypoventilation, which can lead tohypercapnia, pulmonary hypertension, and heart failure. Severe episodichypoxia, which can cause arrhythmias and sudden death, is 10 times morecommon in the severely obese. Severely obese individuals are also atincreased risk of suffering from obstructive sleep apnea, pickwickiansyndrome (i.e., daytime hypoventilation, somnolence, polycythemia, corpulmonale), and renal vein thrombosis. (“Cecil Essentials of Medicine”,Andreoli et al., Third Edition, 1993, W. B. Saunders Company).

[0004] Moderate obesity (corresponding to a weight between 20-60% abovenormal weight) poses increased risk of early mortality. Obeseindividuals suffer more frequently than non obese individuals fromhypertension. Type II diabetes mellitus can also be aggravated by excessweight. Obesity can also increase the risk of a subject developingcholelithiasis and endometrial carcinoma.

[0005] The risk of dyslipidemia and CHD is much greater in bothnon-insulin-dependent (type 2) diabetes mellitus (T2DM) patients andrelatives of T2DM patients compared to non-diabetics (Groop L, et al.(1996) Diabetes 45(11):1585-93; Shaw J T, et al. (1999) Diabetologia42(1):24-27. Among diabetics, the predominant dyslipidemia consists oflow levels of high-density lipoprotein cholesterol (HDL) and hightriglycerides (TG). (Knudsen P, et al. (1995) Diabetologia 38(3):344-50;Ginsberg H N (1996) Diabetes 45(suppl 3):S27-S31; Ginsberg H N (1991)Diabetes Care;14:839-855; Semenkovich D F, Heinecke J W (1997) Diabetes46:327-34).

[0006] One candidate factor that is likely to be involved both inobesity and cardiovascular disease is the SR-BI receptor, which has beenshown to bind HDL and LDL cholesterol and mediate uptake into cells(Acton, S. et al., (1996) Science 271:518-520). SR-BI is likely tocontribute to genetic lipoprotein variability, thereby playing a role inthe development of lipid metabolism disorders and thus generally,cardiovascular diseases.

[0007] In addition, cholesterol gallstone formation could be caused by adefective SR-BI receptor, since the SR-BI receptor is likely to beinvolved in transferring HDL cholesterol from extrahepatic tissues tothe liver (reverse cholesterol transport) e.g. for incorporation intobile (J. L. Breslow, in The Metabolic and Molecular Bases of InheritedDisease, C. R. Scriver, A. L. Beaudet, W. Sly, D. Valle, Eds.(McGraw-Hill, N.Y., 1995), pp 2031-2052; S. M. Grundy, (1995) J Am. Med.Assoc. 256: 2849; G. Assman, A. von Eckardstein, H. B. Brewer Jr. in TheMetabolic and Molecular Bases oflnherited Disease, C. R. Scriver, A. L.Beaudet, W. Sly, D. Valle, Eds. (McGraw-Hill, N.Y., 1995), pp 2053-2072;W. J. Johnson et al., (1991) Biochem. Biophys. Acta 1085:273; M. N.Pieters et al., (1994) Ibid 1225:125; and C. J. Fielding and P. E.Fielding, (1995) J Lipid Res 36:211).

[0008] Further, a defective SR-BI receptor or abnormal levels of SR-BIreceptor could influence the fertility of a subject, since SR-BI appearsto be involved in HDL cholesteryl ester delivery to steroidogenictissues (ovary, adrenal glands and testis) for hormone synthesis (Acton,S. et al., (1996) Science 271:518-520; Landschulz, et al., (1996) JClin. Invest. 98:984-95; J. M. Anderson and J. M. Dietschy (1981) JBiol. Chem. 256: 7362; M. S. Brown et al., (1979) Recent Prog Horm. Res.35:215; J. T. Gwynne and J. F. Strauss III, (1982) Endocr. Rev. 3:299;B. D. Murphy et al., (1985) Endocrinology 116:1587).

[0009] The SR-BI receptor (Scavenger Receptor-BI) is a scavengerreceptor that mediates endocytosis of unmodified and modifiedlipoproteins, e.g., LDL, acetylated LDL, oxidized LDL (Acton et al.(1994) J. Biol. Chem. 269:21003), HDL ((Acton, S. et al., (1996) Science271:518-520), anionic phospholipids (Rigotti et al. (1995) J. Biol.Chem. 270:16221), negatively charged liposomes and apoptotic cells(Fukasawa et al. (1996) Exp. Cell Res. 222:246). The human SR-BIreceptor (also termed “CLA-1) exists in two differentially spliced forms(Calvo and Vega (1993) J. Biol. Chem. 268:18929). The predominant formof human SR-BI is a protein of 509 amino acids. The shorter form of theSR-BI receptor has 409 amino acids, and is lacking the 100 amino acidslocated 42 amino acids downstream of the initiation codon (Calvo andVega, supra). The nucleotide sequence of a cDNA encoding human SR-BI isdisclosed in Calvo and Vega, supra and the nucleotide sequence of a cDNAencoding hamster SR-BI is disclosed in Acton et al. (1994) J. Biol.Chem. 269:21003 and in PCT Application WO 96/00288.

SUMMARY OF THE INVENTION

[0010] The present invention is based at least in part on the discoveryof the genomic structure of the human SR-BI gene and on theidentification of polymorphic regions within the gene, which areassociated with specific diseases or disorders, including abnormnal bodymass and abnormal lipoprotein levels, i.e., low HDL and high LDL levels.Furthermore, the present invention is based, also in part, on thediscovery of significant associations between two common polymorphismswithin the SR-B1 gene and low levels in three ethnically diversepopulations. Furthermore, the associations between SR-B1 polymorphismsand low HDL are influenced by gender, which implies an interaction withhormonal status. Therefore, these SR-B1 polymorphisms may be useful inpredicting the effect of hormone replacement therapy (HRT) on HDL levelsin female subjects, e.g., postmenopausal female subjects.

[0011] The human SR-BI gene contains 12 coding exons and one non codingexon (exon 13). The structure of the gene and the position of theintrons relative to the nucleotide sequence of the exons are shown inFIGS. 1, 2, and 3.

[0012] Several polymorphic regions that are associated with specificdiseases or disorders, have been found in the human SR-BI gene byanalyzing the DNA of a specific population of individuals. Onepolymorphism found in the population is a change from a guanine to anadenine at position 146 in exon 1, which results in a change from aglycine to a serine at amino acid residue 2 of the encoded protein. Asecond polymorphism is a change from a guanine to an adenine at position119 in exon 3, which results in a change from a valine to an isoleucineat amino acid residue 135 of the encoded protein. A third polymorphismis a change from a cytidine to a thymidine at position 41 of exon 8,referred to herein as “EX8C” where there is a cytidine at position 41 ofexon 8 (the more common allele) or “EX8T,” where there is a thymidine atposition 41 of exon 8 (the less common allele). The change from acytidine to a thymidine at position 41 of exon 8 does not result in adifference in the amino acid sequence of the encoded protein. A fourthpolymorphism is a change from a cytidine to a thymidine at position 54of intron 5, referred to herein as “IVS5C” where there is a cytidine atposition 54 of intron 5 (the more common allele) or “IVS5T” where thereis a thymidine at position 54 of intron 5 (the less common allele). Afifth polymorphism is a change from a cytidine to a guanine at position−41 of intron 10 (position −1 corresponds to the first nucleotideupstream of exon 11).

[0013] Specific allelic variants of these polymorphic regions are shownherein to be associated with specific disorders. In particular, thepresence of a thymidine at position 41 in exon 8 (EX8T) was found to beassociated with low plasma LDL levels in women and a thymidine atposition 54 of intron 5 (IVS5T) was found to be associated with a highBMI and high plasma LDL levels in women. In men, the presence of athymidine at position 41 (EX8T) in exon 8, a thymidine at position 54 ofintron 5 (IVS5T) and/or an adenine at position 146 of exon 1 was foundto be associated with a high plasma HDL level. In addition, the presenceof a cytidine at position 41 in exon 8 (EX8C) was found to be associatedwith low HDL levels in both women and men in three ethnically distinctpopulations. The presence of a thymidine at position 54 of intron 5(IVS5T) was found to be associated with low HDL in women in threeethnically distinct populations. Furthermore, in women, the presence ofboth a cytidine at position 41 in exon 8 (EX8C) and a thymidine atposition 54 of intron 5 (IVS5T) was found to be associated with afour-fold increase in the odds of having low HDL across three ethnicallydistinct populations. Since abnormal lipid, lipoprotein levels, and BMImay be associated with obesity, cachexia, diabetes, cardiovasculardisease, gallstone formation and other disorders, SR-BI polymorphicvariants are directly or indirectly associated with obesity, cachexia,diabetes, cardiovascular disease, gallstone formation and otherdisorders. SR-B1 variants, e.g., EX8 and IVS5, may be used to predictthe effect of hormone replacement therapy (HRT) on HDL levels in femalesubjects, e.g., postmenopausal female subjects.

[0014] In one embodiment, the invention provides isolated nucleic acidscomprising an intronic sequence from an SR-BI gene. In a preferredembodiment, the SR-BI gene is a human gene. In another preferredembodiment, the nucleic acid of the invention has a nucleotide sequenceset forth in FIG. 2A-G or in any of the intronic sequences in SEQ IDNos. 1-121, complements thereof, or homologues thereof. In yet anotherembodiment, the intronic sequence of the nucleic acid is capable ofhybridizing under an appropriate stringency to a nucleic acid having anintronic nucleotide sequence set forth in any of SEQ ID Nos. 1-121 orcomplements thereof.

[0015] Other preferred nucleic acids of the invention comprise at leastan allelic variant of a polymorphic region. A preferred allele has apolymorphic region that is located in an exon and comprises, e.g., anucleotide sequence set forth in SEQ ID NO: 65, SEQ ID NO: 95, or SEQ IDNO: 96 or a polymorphic region that is located in an intron andcomprises, e.g., a nucleotide sequence set forth in SEQ ID NO: 66 or SEQID NO: 97. The isolated nucleic acid preferably comprises from about 15to about 30 nucleotides and can comprise, e.g., a nucleotide sequenceselected from the group consisting of SEQ ID NO: 41 to SEQ ID NO: 64 andSEQ ID NO: 89 to SEQ ID NO: 94. The isolated nucleic acid can be doublestranded or single stranded and can further comprise a label.

[0016] The nucleic acids of the invention can be used, e.g., inprognostic, diagnostic, and therapeutic methods. For example, thenucleic acids of the invention can be used as probes or primers todetermine whether a subject has or is at risk of developing a disease ordisorder associated with a specific allelic variant of an SR-BIpolymorphism, e.g., a disease or disorder associated with an aberrantSR-BI activity, e.g, obesity, diabetes, or cardiovascular disease.

[0017] The invention further describes vectors comprised of the claimednucleic acids; host cells transfected with said vectors whetherprokaryotic or eukaryotic; and transgenic non-human animals whichcontain a heterologous form of a functional or non-functional SR-BIallele described herein. Such a transgenic animal can serve as an animalmodel for studying, e.g., the effect of specific allelic variations,including mutations of an SR-BI gene or for use in drug screening orrecombinant protein production.

[0018] The invention further provides methods for determining themolecular structure of at least a portion of an SR-BI gene. In apreferred embodiment, the method comprises contacting a sample nucleicacid comprising an SR-BI gene sequence with a probe or primer having asequence which is complementary to an SR-BI gene sequence and comparingthe molecular structure of the sample nucleic acid with the molecularstructure of a control (known) SR-BI gene (e.g., an SR-BI gene from ahuman not afflicted with a cardiovascular condition or a diseaseassociated with an aberrant SR-BI activity). The method of the inventioncan be used for example in determining the molecular structure of atleast a portion of an exon, an intron, or the promoter. In a preferredembodiment, the method comprises determining the identity of at leastone nucleotide. In even more preferred embodiments, the nucleotide isnucleotide 146 of exon 1, nucleotide 119 of exon 3, nucleotide 41 ofexon 8, nucleotide 54 of intron 5, and/or nucleotide −41 of intron 10.In another preferred embodiment, the method comprises determining thenucleotide content of at least a portion of an SR-BI gene, such as bysequence analysis. In yet another embodiment, determining the molecularstructure of at least a portion of an SR-BI gene is carried out bysingle-stranded conformation polymorphism. In yet another embodiment,the method is an oligonucleotide ligation assay. Other methods withinthe scope of the invention for determining the molecular structure of atleast a portion of an SR-BI gene include hybridization ofallele-specific oligonucleotides, sequence specific amplification, andprimer specific extension.

[0019] In at least some of the methods of the invention, the probe orprimer is allele specific. Preferred probes or primers are singlestranded nucleic acids, which optionally are labeled.

[0020] The methods of the invention can be used for determining theidentity of the allelic variant of a polymorphic region of a human SR-BIgene present in a subject. For example, the method of the invention canbe useful for determining whether a subject has, or is at risk ofdeveloping, a disease or condition associated with a specific allelicvariant of a polymorphic region in the human SR-BI gene. In oneembodiment, the disease or condition is characterized by an aberrantSR-BI activity, such as an aberrant SR-BI protein level, which canresult from an aberrant expression of an SR-BI gene. The disease orcondition can be an abnormal lipid metabolism, inappropriate lipid orlipoprotein level, an abnormal body mass index, atherosclerosis, orgallstone formation. Accordingly, the invention provides methods forpredicting or diagnosing cardiovascular diseases or diabetes, and otherdiseases associated with an aberrant SR-BI activity.

[0021] For example, a female subject having the more common allele (acytidine) at residue 41 of exon 8 of SR-BI (EX8C) has or is likely tohave a tendency of having or developing higher LDL levels than a femalesubject having a thymidine at that position, thereby being at a higherrisk of developing a cardiovascular disease. A female subject having theless common allele (a thymidine) at residue 54 of intron 5 (IVS5T) hasor is likely to have or to develop a high BMI and/or high LDL levels,relative to a female subject having a cytidine at that position; and amale subject having the more common allele at residue 41 of exon 8(EX8C), the more common allele at residue 54 of intron 5 (IVS5C), andthe more common allele at residue 146 of exon 1 is likely to have or todevelop lower HDL levels relative to a subject male having the lesscommon alleles of at least one of these polymorphic regions, therebybeing at a higher risk of developing a cardiovascular disease.Furthermore, a female or a male subject with the more common allele(cytidine) at residue 41 of exon 8 of SR-BI (EX8C) has or is likely tohave a tendency of having or developing lower HDL levels than a femaleor a male subject having the less common allele (thymidine) at thatposition (EX8T), thereby being at a higher risk of developing acardiovascular disease. A female subject with the less common allele(thymidine) at residue 54 of intron 5 (IVS5T) has or is likely to haveor develop low HDL levels, relative to a female subject having the morecommon allele (cytidine) at that position (IVS5C), thereby being at ahigher risk of developing a cardiovascular disease; a female subjecthaving both EX8C and IVS5T has greater than four-fold increased odds ofhaving or developing low HDL levels as compared to a female subjecthaving EX8T and IVS5C, thereby being at a higher risk of developing acardiovascular disease.

[0022] The methods of the invention can also be used in selecting theappropriate drug to administer to a subject to treat a disease orcondition, such as an abnormal lipid metabolism, inappropriate lipidlevel, a cardiovascular disease such as atherosclerosis, gallstoneformation, diabetes, or an abnormal body mass index. In fact, specificallelic variants of SR-BI polymorphic regions may be associated with aspecific response of an individual having such an allele to a specificdrug. For example, a specific SR-BI allele may encode an SR-BI proteinhaving a modified affinity for certain types of molecules, e.g, lipids.Accordingly, the action of a drug necessitating interaction with anSR-BI protein will be different in individuals carrying such an SR-BIallele.

[0023] In a further embodiment, the invention provides a method fortreating a subject having a disease or condition associated with aspecific allelic variant of a polymorphic region of an SR-BI gene. Inone embodiment, the method comprises (a) determining the identity of theallelic variant; and (b) administering to the subject a compound thatcompensates for the effect of the specific allelic variant. In apreferred embodiment, the specific allelic variant is a mutation. Themutation can be located, e.g, in a promoter region, an intron, or anexon of the gene. In one embodiment, the compound modulates (i.e.,agonizes or antagonizes) SR-BI protein levels. In a preferredembodiment, the compound is selected from the group consisting of anucleic acid, a protein, a peptidomimetic, or a small molecule. Thecompound can be, for example, an SR-BI protein. Thus, e.g., if a femalesubject has the more common allele of residue 41 of exon 8 (EX8C), highLDL levels and resulting cardiovascular disorders can be treated,prevented from occurring or can be reduced, by administering to thesubject a pharmaceutically effective amount of a compound which reducesLDL level to a normal LDL level. Similarly, if a female subject has theless common allele of residue 54 of intron 5 (IVS5T), a high BMI and/orLDL level and consequences thereof, such as diabetes and cardiovasculardisorders, can be treated, prevented from occurring or can be reduced,by administering to the subject a pharmaceutically effective amount of acompound which reduces the BMI and/or the LDL levels. If, on the otherhand, a male subject has the more common allele at residue 41 of exon 8(EX8C), the more common allele at residue 54 of intron 5 (IVS5C), andthe more common allele at residue 146 of exon 1, development of low HDLlevels can be treated, prevented, or increased by administering to thesubject a pharmaceutically effective amount of a compound that increasesHDL levels to normal levels. Likewise, if a female or a male subject hasthe more common allele at residue 41 of exon 8 (EX8C), development oflow HDL levels and resulting cardiovascular disorders can be treated orprevented from occurring by administering to the subject apharmaceutically effective amount of a compound which increases HDLlevel to normal HDL levels. Similarly, if a female subject has the lesscommon allele of residue 54 of intron 5 (IVS5T), low HDL and resultingcardiovascular disorders can be treated or prevented from occurring, byadministering to the subject a pharmaceutically effective amount of acompound which increases HDL level to a normal HDL level. Furthermore,if a female subject has both the more common allele at residue 41 ofexon 8 (EX8C) and the less common allele of residue 54 of intron 5(IVS5T), low HDL levels and resulting cardiovascular disorders can betreated or prevented from occurring, by administering to the subject apharmaceutically effective amount of a compound which increases HDLlevel to a normal HDL level.

[0024] Since the effect of the presence of the variants are influencedby gender, the identification of one or more SR-B1 variants in a subjectmay be used to predict the effect of hormone replacement therapy (HRT).For example, if a female subject has one or more SR-B1 variants whichhave been associated with low HDL (e.g., EX8C or IVS5T), but has normalHDL, HRT may cause low HDL in that subject. Likewise, if a femalesubject does not have either variant associated with low HDL (e.g., EX8Cor IVS5T), then it can be predicted that HRT would not cause low HDL inthat subject. Therefore, in another embodiment, the invention provides amethod of predicting the effect of HRT on a female subject with normalHDL levels, wherein the identification of allelic variants of the SR-B1gene which are associated with abnormally low HDL levels in femalesresults in a prediction that HRT will result in abnormally low HDLlevels.

[0025] The invention also provides probes and primers comprisingsubstantially purified oligonucleotides, which correspond to a region ofnucleotide sequence which hybridizes to at least 6 consecutivenucleotides of the sequence set forth as SEQ ID Nos: 1, 2, or 3 or tothe complement of the sequences set forth as SEQ ID Nos: 1, 2, or 3; ornaturally occurring mutants thereof. In preferred embodiments, theprobe/primer further includes a label group attached thereto, which iscapable of being detected.

[0026] In another embodiment, the invention provides a kit foramplifying and/or for determining the molecular structure of at least aportion of an SR-BI gene, comprising a probe or primer capable ofhybridizing to an SR-BI gene and instructions for use. In oneembodiment, the probe or primer is capable of hybridizing to an SR-BIintron. In another embodiment, the probe or primer is capable ofhybridizing to an allelic variant of a polymorphic region. In apreferred embodiment, the polymorphic region is located in an exon, suchas exon 1, 3, or 8 or in an intron, such as intron 5 or 10. In apreferred embodiment, determining the molecular structure of a region ofan SR-BI gene comprises determining the identity of the allelic variantof the polymorphic region. Determining the molecular structure of atleast a portion of an SR-BI gene can comprise determining the identityof at least one nucleotide or determining the nucleotide composition,e.g., the nucleotide sequence.

[0027] A kit of the invention can be used, e.g., for determining whethera subject has or is at risk of developing a disease associated with aspecific allelic variant of a polymorphic region of an SR-BI gene, e.g.,EX8C or IVS5T. In a preferred embodiment, the invention provides a kitfor determining whether a subject has or is at risk of developing adisease or condition associated with abnormal lipid metabolism,inappropriate lipid or lipoprotein levels, a cardiovascular disease suchas atherosclerosis, gallstone formation, diabetes, or an abnormal bodymass index. The disease or condition can be associated with an aberrantSR-BI activity, which can result, e.g., from a mutation in the SR-BIgene. The kit of the invention can also be used in selecting theappropriate drug to administer to a subject to treat a disease orcondition, such as a disease or condition set forth above. In fact,pharmacogenetic studies have shown that the genetic background ofindividuals play a role in determining the response of an individual toa specific drug. Thus, determining the allelic variants of SR-BIpolymorphic regions of an individual can be useful in predicting how anindividual will respond to a specific drug, e.g., a drug for treating adisease or disorder associated with an aberrant SR-BI activity and/or acardiovascular disease or a disease associated with an aberrant lipidlevel.

[0028] Other features and advantages of the invention will be apparentfrom the following detailed description and claims.

BRIEF DESCRIPTION OF THE FIGURES

[0029]FIG. 1 is a schematic depiction of the chromosomal structure ofthe human SR-BI gene indicating the introns (1 through 12) and exons(I-XIII). Black boxes represent coding exons (exons I-XII) and the whitebox represents the non-coding exon (exon XIII) boxed and the nucleotidesin the newly identified alleles are indicated.

[0030]FIG. 2A-G represents the nucleotide sequence of the exons(underlined sequence) of the human SR-BI gene, portions of the intronswhich are adjacent to the exons, and 3′ end of the promoter sequence(SEQ ID Nos. 5-40). The putative 5′ end of the cDNA, as predicted byGRAIL is indicated in italics. The TATA-like box is indicated in italicsand is boxed. Bold sequences correspond to the nucleotide sequence orthe complement of the nucleotide sequence of preferred primers foramplifying each of the exons or a promoter region. The nucleotidepolymorphisms in exons 1, 3, and 8 and introns 5 and 10 are boxed.

[0031]FIG. 3A-B shows the nucleotide sequence of the full length humanSR-BI cDNA (SEQ ID NO: 1) and the position of introns 1-12 relative tothe nucleotide sequence of the exons. The nucleotide polymorphisms inexons 1, 3, and 8 are boxed.

[0032]FIG. 4 is a graphic representing the mean LDL-C differences(+/−95% Confidence intervals) between SR-BI genotypes carrying variantalleles and the wild-type genotype (111/111) in women. *significantlydifferent from 111/111 (p<0.030). The differences between genotypessharing letters are statistically significant (a: p=0.001; b: p=0.016;c: p=0.004).

[0033]FIG. 5 is a graphic representing the mean LDL-C differences(+/−95% Confidence intervals) between SR-BI genotypes carrying variantalleles and the wild-type genotype (111/111) in men. *significantlydifferent from 111/111 (p<0.030). The differences between genotypessharing letters are statistically significant (a: p=0.001; b: p=0.016;c: p=0.004).

[0034]FIG. 6 is a graphic representing mean HDL differences (+/−95%Confidence intervals) between SR-BI genotypes carrying variant allelesand the wild-type genotype (111/111) in women.

[0035]FIG. 7 is a graphic representing mean HDL differences (+/−95%Confidence intervals) between SR-BI genotypes carrying variant allelesand the wild-type genotype (111/111) in men. *significantly differentfrom 111/111 (p<0.040).

[0036]FIG. 8 is a graphic representing mean BMI differences (+/−95%Confidence intervals) between SR-BI genotypes carrying variant allelesand the wild-type genotype (111/111) in women. *significantly differentfrom 111/111 (p=0.020). The differences between genotypes sharingletters are statistically significant (a: p=0.007; b: p=0.005; c:p=0.004).

[0037]FIG. 9 is a graphic representing mean BMI differences (+/−95%Confidence intervals) between SR-BI genotypes carrying variant allelesand the wild-type genotype (111/111) in men.

DETAILED DESCRIPTION OF THE INVENTION

[0038] General

[0039] The present invention is based at least in part on the discoveryof the genomic structure of the human SR-BI gene and on theidentification of polymorphic regions within the gene which correlatewith specific diseases or conditions, such as an abnormal BMI or anabnormal lipoprotein (i.e., HDL or LDL) level. The present invention isbased, at least in part, on the on the discovery of significantassociations between two common polymorphisms, e.g., position 41 of exon8 (EX8) and position 54 of intron 5 (IVS5), within the SR-B1 gene andlow HDL cholesterol (HDL) in three ethnically diverse populations. Thethree ethnically diverse populations consisted of two Scandanavianpopulations, one from Finland and one from southern Sweden, and a thirdpopulation of Israelis of Ashkenazi Jewish origin. These subjects weredrawn from collections of nuclear families ascertained from non-insulindependent (type-2) diabetes mellitus (T2DM).

[0040] Both EX8C (e.g., the more common allele at residue 41 of exon 8),and IVS5T (e.g., the less common allele at residue 54 of intron 5) alonewere positively associated with low HDL in female subjects in all threepopulations. In male subjects, the presence of EX8C alone was shown tohave a positive association with low HDL across all three populations.For male subjects, the presence of IVS5T alone was inversely associatedwith low HDL levels. The associations between SR-B1 EX8 and IVS5 allelesand low HDL levels are presented in Table I for women and Table II formen, set forth below. TABLE I Association between SR-B1 EX8 and IVS5status and low HDL in women. IVS5T EX8C carriers carriers Con- P Con- PPopulation Cases trols OR* value Cases trols OR value Ashkenazi 79% 59%18% 10% unadjusted 2.59 .003  2.06 .11  adjusted 2.50 .008  2.42 .065Finnish 88% 71% 10%  2% unadjusted 2.92 .013  4.95 .13  adjusted† 3.69.015  3.33 .28  Swedish 83% 68% 33% 13% unadjusted 2.33 .054  3.34 .013adjusted 2.75 .044  2.73 .069 Combined 83% 65% 18%  9% unadjusted 2.66<.00001 2.36 .006 adjusted 2.84 <.0001  2.32 .010

[0041] TABLE II Association between SR-B1 EX8 and IVS5 status and lowHDL in men. IVS5T EX8C carriers carriers Con- P Con- P Population Casestrols OR* value Cases trols OR value Ashkenazi 75% 61% 17% 17%unadjusted 1.95 .048 0.97 .95  adjusted† 2.11 .038 1.31 .55  Finnish 88%85%  4% 10% unadjusted 1.38 .50  0.37 .16  adjusted 1.54 .42  0.43 .30 Swedish 84% 64%  8% 23% unadjusted 2.82 .014 0.30 .024 adjusted 3.02.020 0.25 .020 Combined 81% 71% 11% 16% unadjusted 1.81 .008 0.62 .084adjusted 1.79 .015 0.64 .15 

[0042] Among female subjects, the presence of both EX8C and IVS5Tresults in four-fold increased odds of having or developing low HDL ascompared to those without either variant. Male carriers of both EX8C andIVS5T, were no more likely to have low HDL than individuals withouteither variant. The combined effect of both variants on HDL level isshown in Table III, below. TABLE III Effect of both EX8 and IVS5 statuson low HDL in female and male subjects from the three populationscombined. EX8C IVS5T cases controls OR 95% C.I. P value WOMEN + + 53 154.79 (2.30, 10.07) <.00001 + − 189 105 2.44 (1.53, 3.89)  <.0001  − −/+*48 65 1.00 — — chi square p < 2 × 10⁻⁶ MEN + + 28 29 1.08 (0.54, 2.18) .95  + − 184 106 1.95 (1.21, 3.14)  <.01   − −/+† 49 55 1.00 — — chisquare p < .006

[0043] As described above, female carriers of both EX8C and IVS5T hadthe highest odds of having low HDL. However, even carriers of EX8C wholacked the IVS5T variant were at increased odds of having low HDL.Considering the nature of these variants (EX8 is silent and IVS5intronic), the results suggest that neither EX8C nor IVS5T are thecausative polymorphism underlying the association with low HDL. Theunderlying variant(s) may be found within haplotypes represented bydifferent combinations of alleles at both EX8 and IVS5. As used herein,a “haplotype” is a set or pattern of polymorphisms which are conservedand which confer a certain phenotype. Two different risk patterns areevident in women. The first pattern, conferring the greater thanfour-fold increased odds of low HDL, is defined by presence of both EX8Cand IVS5T. The second pattern, conferring increased (>2.4-fold) odds oflow HDL, is defined by presence of EX8C and absence of IVS5T. In men,only the second pattern was associated with low HDL, conferring about atwo-fold increased odds. This association is consistent with what wasfound in women. However, unlike women, men with the first pattern wereno more likely to have low HDL than those who lacked both variantalleles.

[0044] It is well known that HDL levels are affected by sex hormonestatus. Furthermore, the expression of SR-B1 is known to be regulated byestrogen. Estrogen treatment of rats has been shown to downregulateSR-B1 in the liver (Fluiter K, et al. (1998) J Biol Chem. 273:8434-8).Moreover, overexpression of SR-B1 in the liver has been demonstrated toresult in a pronounced fall in plasma HDL (Kozarsky KF (1997) Nature387:414-7). It is possible that the downregulation of SR-B1 by estrogenis impaired by a genetic variant in SR-B1, resulting in an increasedexpression of SR-B1 and therefore lower HDL levels in women. This sameeffect may not be apparent in men as estrogen does not play as key arole in the modulation of HDL.

[0045] The possible interaction between the SR-B1 variants and hormonalstatus has implications for the treatment of females with hormonereplacement therapy (HRT). It is possible that SR-B1 variants maymodulate the effect of HRT on HDL levels in women. For example, apostmenopausal woman may have the EX8C and IVS5T variants, but may havenormal HDL. However, treatment with HRT may cause low HDL. Therefore, infemales, the identification of SR-B1 variants (e.g., EX8C and/or IVS5T)may be used to predict the effect HRT would have on HDL level (e.g.,lowering HDL level). SR-B1 genotype may also have utility as apharmacogenomic marker of response to lipid lowering therapies.

[0046] In women, the associations between SR-B1 variants and low HDLwere consistently demonstrated across three different ethnicpopulations. In men, some population-specific effects were observed. Theability to reproduce an association in multiple populations adds to thevalidity of these results. It is unlikely that the observed associationswere spurious due to population stratification in all three of theserelatively homogenous groups. The replication of these associations inthree distinct ethnic groups also suggests that the haplotypes carryingthe underlying variants may be the same across populations.

[0047] The strongest association with HDL level is not with eithervariant alone, but rather the combination of genotypes at EX8 and IVS5.Therefore, the location of additional genetic variants in or around theSR-B1 locus, in linkage disequilibrium with IVS5 and EX8, may influencethese associations. These additional genetic variants may include thevariants described herein or other variants present within the SR-B1gene.

[0048] The association of SR-B1 polymorphisms with low HDL remainedsignificant after controlling for other covariates known to beassociated with HDL including T2DM status, BMI, age and triglyceride(TG) levels. This suggests that SR-B1 is an independent predictor of HDLlevels. Most of the individuals were affected or related to individualsaffected with T2DM. Combined low HDL and high TG are the hallmarkdyslipidemia associated with T2DM. This combined phenotype may be areasonable marker of insulin resistance (Jeppesen, et al (1997) JArterioscler Thromb Vasc Biol. 17:1114-20).

[0049] SR-B1 genetic variants showed strong associations with low HDL.The high frequency of the variants combined with their strong effectsuggests that SR-B1 is a major genetic determinant of low HDL. Thefraction of low HDL attributable to the EX8 variant was estimated to be35% in women and 23% in men in the three populations tested. Theassociation of SR-B1 variants with low HDL described herein is not onlystrong but highly significant and reproducible. Therefore, SR-B1 may beused as a genetic marker of plasma HDL cholesterol levels.

[0050] As shown in FIG. 1, the human SR-BI gene is at least 50 kilobasepairs long and has 12 coding exons, one non-coding exon (exon 13), and12 introns. The exons are numbered 1 to 13 from 5′ to 3′ and the intronsare labeled 1 through 12 from 5′ to 3′ . Exon 1 corresponds to the firstexon located downstream of the promoter and contains the initiationcodon. Intron 1 is located immediately downstream of exon I (see FIG.1). The position of the introns relative to the nucleotide sequence ofthe full length cDNA encoding SR-BI is shown in FIG. 2A-G. Thenucleotide sequence of the human SR-BI cDNA, shown in FIG. 3 and in SEQID NO: 1 encodes a protein of 509 amino acids. SEQ ID NO. 1 contains thenucleotide sequence of the cDNA disclosed in Calvo and Vega (1993) J.Biol. Chem. 268:18929, and contains in addition a complete 5′ end. Theamino acid sequence of the protein set forth in SEQ ID NO: 2 isidentical to the Cla-I protein disclosed in Calvo and Vega (1993) J.Biol. Chem. 268:18929. As set forth in Calvo and Vega, supra,differential splicing of the human SR-BI gene also results in a shortMRNA lacking 300 nucleotides located 126 nucleotides downstream of theinitiation codon, i.e., lacking exons 2 and 3 set forth in FIG. 3, whichencodes a protein of 409 amino acids. The shorter protein is referred toherein as “splice variant”. The nucleotide sequence of a full lengthcDNA encoding the splice variant is set forth in SEQ ID NO: 3 and theamino acid sequence of the SR-BI splice variant protein encoded by thisnucleotide sequence is set forth in SEQ ID NO: 4. The splice variant israre relative to the 509 amino acid SR-BI protein.

[0051]FIG. 2A-G shows the nucleotide sequence of the 3′ end of the SR-BIpromoter. Additional promoter sequence is disclosed in U.S. patentapplication 08/812,204 by Acton, incorporated herein by reference.

[0052] Set forth below in Table IV are the locations and sizes of theexons in the human SR-BI gene relative to the nucleotide sequence of afull length cDNA encoding human SR-BI protein (SEQ ID NO: 1), in whichnucleotide 1 corresponds to the first nucleotide in the isolatedtranscript. Table IV also indicates the portions of the human SR-BIprotein encoded by each of these exons. Amino acid 1 is the initiatingmethionine. Also indicated is the length of the intron locateddownstream of each of the exons. TABLE IV cDNA Nucleotide Amino acidposition position size of intron Exon 1  1-244  1-42 intron 1: >2827Exon 2 245-402 43-95 intron 2: 2429 Exon 3 403-544  95-142 intron 3: 567Exon 4 545-748 143-210 intron 4: 2229 Exon 5 749-844 211-242 intron 5:1580 Exon 6 845-960 243-281 intron 6: >10532 Exon 7  961-1127 281-337intron 7: >3985 Exon 8 1228-1246 337-376 intron 8: >11321 Exon 91247-1320 377-401 intron 9: 7562 Exon 10 1321-1372 401-418 intron 10:902 Exon 11 1373-1519 419-467 intron 11: 3547 Exon 12 1520-1648 468-509intron 12: >4578 Exon 13 1649-2630

[0053]FIG. 2A-G shows the nucleotide sequence of portions of the intronswhich are adjacent to the exons. The nucleotide sequence of each of theexons and adjacent portions of introns shown in FIG. 2A-G are set forthin SEQ ID Nos. 5 to 16. The portions of each of the introns shown inFIG. 2A-G are set forth in SEQ ID Nos. 18 to 40. For convenience, theidentity of the sequences referred to as SEQ ID Nos. 1 to 40 are setforth below in Table V: TABLE V SEQ ID NO: 1 full length cDNA encodinghuman SR-BI; SEQ ID NO: 2 full length amino acid sequence of human SR-BIprotein; SEQ ID NO: 3 full length cDNA encoding splice variant of humanSR-BI (Calvo and Vega, supra); SEQ ID NO: 4 full length amino acidsequence of splice variant of human SR-BI protein (Calvo and Vega,supra); SEQ ID NO: 5 3′ end of promoter, exon 1, and 5′ end of intron 1;SEQ ID NO: 6 3′ end of intron 1, exon 2, and 5′ end of intron 2; SEQ IDNO: 7 3′ end of intron 2, exon 3, and 5′ end of intron 3; SEQ ID NO: 83′ end of intron 3, exon 4, and 5′ end of intron 4; SEQ ID NO: 9 3′ endof intron 4, exon 5, and 5′ end of intron 5; SEQ ID NO: 10 3′ end ofintron 5, exon 6, and 5′ end of intron 6; SEQ ID NO: 11 3′ end of intron6, exon 7, and 5′ end of intron 7; SEQ ID NO: 12 3′ end of intron 7,exon 8, and 5′ end of intron 8; SEQ ID NO: 13 3′ end of intron 8, exon9, and 5′ end of intron 9; SEQ ID NO: 14 3′ end of intron 9, exon 10,and 5′ end of intron 10; SEQ ID NO: 15 3′ end of intron 10, exon 11, and5′ end of intron 11; SEQ ID NO: 16 3′ end of intron 11, exon 12, and 5′end of intron 12; SEQ ID NO: 17 3′ end of promoter; SEQ ID NO: 18 5′ endof intron 1; SEQ ID NO: 19 3′ end of intron 1; SEQ ID NO: 20 5′ end ofintron 2; SEQ ID NO: 21 3′ end of intron 2; SEQ ID NO: 22 5′ end ofintron 3; SEQ ID NO: 23 3′ end of intron 3; SEQ ID NO: 24 5′ end ofintron 4; SEQ ID NO: 25 3′ end of intron 4; SEQ ID NO: 26 5′ end ofintron 5; SEQ ID NO: 27 3′ end of intron 5; SEQ ID NO: 28 5′ end ofintron 6; SEQ ID NO: 29 3′ end of intron 6; SEQ ID NO: 30 5′ end ofintron 7; SEQ ID NO: 31 3′ end of intron 7; SEQ ID NO: 32 5′ end ofintron 8; SEQ ID NO: 33 3′ end of intron 8; SEQ ID NO: 34 5′ end ofintron 9; SEQ ID NO: 35 3′ end of intron 9; SEQ ID NO: 36 5′ end ofintron 10; SEQ ID NO: 37 3′ end of intron 10; SEQ ID NO: 38 5′ end ofintron 11; SEQ ID NO: 39 3′ end of intron 11; and SEQ ID NO: 40 5′ endof intron 12.

[0054] An analysis of the human SR-BI gene in a population ofindividuals chosen because these individuals had a known age, known HDLand LDL levels, known body mass index, and known triglycerides and totalcholesterol levels revealed the existence of several polymorphisms inthe SR-BI gene in this population. These polymorphisms were identifiedby performing single stranded conformation polymorphism (SSCP) analysisof genomic DNA from independent individuals as described in Example 3and in Example 6, using PCR primers complementary to intronic orpromoter sequences surrounding each of the exons. The nucleotidesequence of these PCR primers (having SEQ ID Nos. 41-64) is shown inTable VII (in the Examples).

[0055] The results indicated the presence of at least five polymorphicregions in the human SR-BI gene in the population studied. The locationand identity of these polymorphisms is indicated in Table VI. TABLE VILocations of polymorphisms in the human SR-BI gene. poly- cDNA aminoacid morphism position position change location exon 1    4   2 Gly−> Ser  1     2     3 ATG (G/A)GC TGC exon 3  403 135 Val −> Ile  135   136 137 (G/A)TC ATG CCC intron 5 na na na 240 241 242 CTG AGCAAG gtgaggggcgagaggcgagggcccctgt             cgccagggaggggagggtgggcc(c/t)g (SEQ ID NO.:87) exon 8 1050350 none   350   351 352 (C/T)GA CCC GGT intron 10 na na na                                               419c(c/g)tgcggccccagctcatgtgtttgtcattctgtctcctcag AGC 420 421 GGG GCC (SEQID NO.:88)

[0056] As can be seen in Table VI, one polymorphism is a change from aguanine to an adenine at position 146 in exon 1, which results in achange from a glycine to a serine at amino acid residue 2 of the encodedprotein. The nucleotide sequence of this allele is set forth in SEQ IDNO: 95 (which is identical to SEQ ID NO: 5, except for nucleotide 146 ofexon 1 which is an adenine). A second polymorphism is a change from aguanine to an adenine at position 119 in exon 3, which results in achange from a valine to an isoleucine at amino acid residue 135 of theencoded protein. The nucleotide sequence of this allele is set forth inSEQ ID NO: 96 (which is identical to SEQ ID NO: 7, except for nucleotide119 of exon 3 which is an adenine).

[0057] A third polymorphism is a change from a cytidine to a thymidineat position 41 of exon 8, which does not result in a difference in theamino acid sequence of the encoded protein. The nucleotide sequence ofexon 8 of this allele is set forth in SEQ ID NO: 65 (SEQ ID NO: 65 isidentical to SEQ ID NO: 12, except for nucleotide 41 of the exonsequence which is a thymidine). About 35% of the individuals of aSpanish population of 142 individuals were found to be homozygous forthe allele having a cytidine at position 41 (i.e., SR-BI sequenceoriginally disclosed); about 17% of the individuals were found to behomozygous for the allele having a thymidine at position 41 of exon 8;and about 48% of the individuals were found to be heterozygous, i.e.,having one allele having a cytidine at position 41 and one allele havinga thymidine at position 41.

[0058] A fourth polymorphism is a change from a cytidine to a thymidineat position 54 of intron 5 (position 1 being defined as the firstnucleotide in the intron). This nucleotide substitution destroys theApal restriction site which is present when the nucleotide at position54 is a cytidine. The nucleotide sequence of the 5′ end of intron 5 ofthis allele is set forth in SEQ ID NO: 66 (SEQ ID NO: 66 is identical toSEQ ID NO: 26, except for nucleotide 54 which is a thymidine).

[0059] A fifth polymorphism in the SR-BI gene is a change from acytidine to a guanine at position −41 of intron 10 (position −1corresponds to the first nucleotide upstream of exon 11). The nucleotidesequence of the 3′ end of intron 10 of this allele is set forth in SEQID NO: 97 (SEQ ID NO: 97 is identical to SEQ ID NO: 15, except fornucleotide −41 of intron 10 which is a guanine).

[0060] Further analysis of the human SR-BI gene is likely to reveal theexistence of yet other polymorphic regions. Such analysis can beperformed using the methods described herein and genomic DNA from randomsubjects or subjects of families associated with specific diseases. Forexample, the polymorphism studies described herein can also be appliedto populations in which cholesterol gallstones are prevalent.Accordingly, the invention provides materials and methods, such asnucleic acids (e.g, intronic sequences useful as probes or primers) fordetermining the identity of other allelic variants of an SR-BIpolymorphic region. The invention also provides methods for determiningthe identity of the alleles of a specific polymorphic region of an SR-BIgene (see e.g., Examples 1-4). Such methods can be used, for example, todetermine whether a subject has or is at risk of developing a disease orcondition associated with one or more specific alleles of polymorphicregions of an SR-BI gene (see e.g., Example 5). In a preferredembodiment, the disease or condition is caused or contributed to by anaberrant SR-BI bioactivity. Other aspects of the invention are describedbelow or will be apparent to one of skill in the art in light of thepresent disclosure.

[0061] 4.2 Definitions

[0062] For convenience, the meaning of certain terms and phrasesemployed in the specification, examples, and appended claims areprovided below.

[0063] The term “allele”, which is used interchangeably herein with“allelic variant” refers to alternative forms of a gene or portionsthereof. Alleles occupy the same locus or position on homologouschromosomes. When a subject has two identical alleles of a gene, thesubject is said to be homozygous for the gene or allele. When a subjecthas two different alleles of a gene, the subject is said to beheterozygous for the gene. Alleles of a specific gene can differ fromeach other in a single nucleotide, or several nucleotides, and caninclude substitutions, deletions, and insertions of nucleotides. Anallele of a gene can also be a form of a gene containing a mutation.

[0064] The term “allelic variant of a polymorphic region of an SR-BIgene” refers to a region of an SR-BI gene having one of severalnucleotide sequences found in that region of the gene in otherindividuals.

[0065] “Biological activity” or “bioactivity” or “activity” or“biological fuinction”, which are used interchangeably, for the purposesherein when applied to SR-BI means an effector or antigenic functionthat is directly or indirectly performed by an SR-BI polypeptide(whether in its native or denatured conformation), or by any subsequence(fragment) thereof. Biological activities include binding to a ligand,e.g., a lipid or lipoprotein, such as LDL or modified forms thereof, orHDL or modified forms thereof. Other molecules which can bind an SR-BIreceptor include anionic molecules, such as anionic phospholipids,negatively charged liposomes, and apoptotic cells. Another biologicalactivity of an SR-BI protein includes endocytosis of a ligandinteracting with the receptor. A biological activity is also intended toinclude binding to a protein, such as binding to the cytoplasmic domainof SR-BI. Yet other biological activities include signal transductionfrom the receptor, modulation of expression of genes responsive tobinding of a ligand to an SR-BI receptor, and other biologicalactivities, whether presently known or inherent. An SR-BI bioactivitycan be modulated by directly affecting an SR-BI protein. Alternatively,an SR-BI bioactivity can be modulated by modulating the level of anSR-BI protein, such as by modulating expression of an SR-BI gene.Antigenic functions include possession of an epitope or antigenic sitethat is capable of cross-reacting with antibodies raised against anaturally occurring or denatured SR-BI polypeptide or fragment thereof.

[0066] Biologically active SR-BI polypeptides include polypeptideshaving both an effector and antigenic function, or only one of suchfunctions. SR-BI polypeptides include antagonist polypeptides and nativeSR-BI polypeptides, provided that such antagonists include an epitope ofa native SR-BI polypeptide. An effector function of SR-BI polypeptidecan be the ability to bind to a ligand, e.g., a lipid or modified formthereof.

[0067] As used herein the term “bioactive fragment of a SR-BI protein”refers to a fragment of a full-length SR-BI protein, wherein thefragment specifically mimics or antagonizes the activity of a wild-typeSR-BI protein. The bioactive fragment preferably is a fragment capableof binding to a second molecule, such as a ligand.

[0068] The term “an aberrant activity” or “abnormal activity”, asapplied to an activity of a protein such as SR-BI, refers to an activitywhich differs from the activity of the wild-type or native protein orwhich differs from the activity of the protein in a healthy subject,e.g., a subject not afflicted with a disease associated with a specificallelic variant of an SR-BI polymorphism. An activity of a protein canbe aberrant because it is stronger than the activity of its nativecounterpart. Alternatively, an activity can be aberrant because it isweaker or absent related to the activity of its native counterpart. Anaberrant activity can also be a change in an activity. For example anaberrant protein can interact with a different protein relative to itsnative counterpart. A cell can have an aberrant SR-BI activity due tooverexpression or underexpression of the gene encoding SR-BI. Anaberrant SR-BI activity can result, e.g., from a mutation in the gene,which results, e.g., in lower or higher binding affinity of a lipid tothe SR-BI protein encoded by the mutated gene. An aberrant SR-BIactivity can also result from a lower or higher level of SR-BI receptoron cells, which can result, e.g., from a mutation in the 5′ flankingregion of the SR-BI gene or any other regulatory element of the SR-BIgene, such as a regulatory element located in an intron. Accordingly, anaberrant SR-BI activity can result from an abnormal SR-BI promoteractivity.

[0069] The terms “abnormal SR-BI promoter activity”, “aberrant SR-BIpromoter activity”, “abnormal SR-BI transcriptional activity” and“aberrant SR-BI transcriptional activity”, which are usedinterchangeably herein, refer to the transcriptional activity of anSR-BI promoter which differs from the transcriptional activity of thesame promoter in a healthy subject. An abnormal SR-BI activity canresult from a higher or lower transcriptional activity than that in ahealthy subject. An aberrant SR-BI promoter activity can result, e.g.,from the presence of a genetic lesion in a regulatory element, such asin a regulatory element located in the promoter. An “aberrant SR-BIpromoter activity” is also intended to refer to the transcriptionalactivity of an SR-BI promoter which is functional (capable of inducingtranscription of a gene to which it is operably linked) in tissues orcells in which the “natural” or wild-type SR-BI promoter is notfunctional or which is non functional in tissues or cells in which the“natural” or wild-type SR-BI promoter is functional. Thus, a tissuedistribution of SR-BI in a subject which differs from the tissuedistribution of SR-BI in a “normal” or “healthy” subject, can be theresult of an abnormal transcriptional activity from the SR-BI promoterregion. Such an abnormal transcriptional activity can result, e.g., fromone or more mutations in a promoter region, such as in a regulatoryelement thereof. An abnormal transcriptional activity can also resultfrom a mutation in a transcription factor involved in the control ofSR-BI gene expression.

[0070] The term “body mass index” or “BMI” refers to the ratio of weight(kg)/height (m)² and can be used to define whether a subject isoverweight. Typically, a subject is underweight if he has a BMI<20;normal if he has a BMI of 20-25, overweight if he has a BMI of 25-30,obese if he has a BMI of 30-40 and severely obese if he has a BMI>40.

[0071] As used herein, a subject has an “abnormal body mass” or“abnormal body mass index” or “aberrant body mass” or “aberrant bodymass index” if his body mass index is outside the range defined for ahealthy or normal subject, i.e., BMI of 20-25. A disorder of body massinclude any disorder affecting the body mass of a subject such that hisbody mass is outside the normal range. For example, obesity is adisorder of body mass. Wasting is also a disorder of body mass. Anabnormal body mass index can have a hormonal origin, e.g., inpremenopausal women.

[0072] The term “cardiovascular disorder” refers to a disease ordisorder of the cardiovascular system and includes ischemia, restenosis,congestive heart failure, and atherosclerosis.

[0073] “Cells,” “host cells” or “recombinant host cells” are terms usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

[0074] As used herein, the term “gene” or “recombinant gene” refers to anucleic acid molecule comprising an open reading frame and including atleast one exon and (optionally) an intron sequence. The term “intron”refers to a DNA sequence present in a given gene which is spliced outduring mRNA maturation.

[0075] “Hormone replacement therapy” (HRT) refers to the administrationof female hormones to a female subject, e.g, a postmenopausal femalesubject.

[0076] “Homology” or “identity” or “similarity” refers to sequencesimilarity between two peptides or between two nucleic acid molecules.Homology can be determined by comparing a position in each sequencewhich may be aligned for purposes of comparison. When a position in thecompared sequence is occupied by the same base or amino acid, then themolecules are homologous at that position. A degree of homology betweensequences is a function of the number of matching or homologouspositions shared by the sequences. An “unrelated” or “non-homologous”sequence shares less than 40% identity, though preferably less than 25%identity, with one of the sequences of the present invention.

[0077] The term “a homologue of a nucleic acid” refers to a nucleic acidhaving a nucleotide sequence having a certain degree of homology withthe nucleotide sequence of the nucleic acid or complement thereof. Ahomologue of a double stranded nucleic acid having SEQ ID NO: x isintended to include nucleic acids having a nucleotide sequence which hasa certain degree of homology with SEQ ID NO: x or with the complementthereof. Preferred homologues of nucleic acids are capable ofhybridizing to the nucleic acid or complement thereof.

[0078] The term “interact” as used herein is meant to include detectableinteractions between molecules, such as can be detected using, forexample, a hybridization assay. The term interact is also meant toinclude “binding” interactions between molecules. Interactions may be,for example, protein-protein, protein-nucleic acid, protein-smallmolecule or small molecule-nucleic acid in nature.

[0079] The term “intronic sequence” or “intronic nucleotide sequence”refers to the nucleotide sequence of an intron or portion thereof.

[0080] The term “isolated” as used herein with respect to nucleic acids,such as DNA or RNA, refers to molecules separated from other DNAs orRNAs, respectively, that are present in the natural source of themacromolecule. The term isolated as used herein also refers to a nucleicacid or peptide that is substantially free of cellular material, viralmaterial, or culture medium when produced by recombinant DNA techniques,or chemical precursors or other chemicals when chemically synthesized.Moreover, an “isolated nucleic acid” is meant to include nucleic acidfragments which are not naturally occurring as fragments and would notbe found in the natural state. The term “isolated” is also used hereinto refer to polypeptides which are isolated from other cellular proteinsand is meant to encompass both purified and recombinant polypeptides.

[0081] The term “lipid” shall refer to a fat or fat-like substance thatis insoluble in polar solvents such as water. The term “lipid” isintended to include true fats (e.g. esters of fatty acids and glycerol);lipids (phospholipids, cerebrosides, waxes); sterols (cholesterol,ergosterol) and lipoproteins (e.g. HDL, LDL and VLDL).

[0082] The term “locus” refers to a specific position in a chromosome.For example, a locus of an SR-BI gene refers to the chromosomal positionof the SR-BI gene.

[0083] The term “modulation” as used herein refers to both upregulation,(i.e., activation or stimulation), for example by agonizing; anddownregulation (i.e. inhibition or suppression), for example byantagonizing of a bioactivity (e.g. expression of a gene).

[0084] The term “molecular structure” of a gene or a portion thereofrefers to the structure as defined by the nucleotide content (includingdeletions, substitutions, additions of one or more nucleotides), thenucleotide sequence, the state of methylation, and/or any othermodification of the gene or portion thereof.

[0085] The term “mutated gene” refers to an allelic form of a gene,which is capable of altering the phenotype of a subject having themutated gene relative to a subject which does not have the mutated gene.If a subject must be homozygous for this mutation to have an alteredphenotype, the mutation is said to be recessive. If one copy of themutated gene is sufficient to alter the genotype of the subject, themutation is said to be dominant. If a subject has one copy of themutated gene and has a phenotype that is intermediate between that of ahomozygous and that of a heterozygous (for that gene) subject, themutation is said to be co-dominant.

[0086] As used herein, the term “nucleic acid” refers to polynucleotidessuch as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleicacid (RNA). The term should also be understood to include, asequivalents, derivatives, variants and analogs of either RNA or DNA madefrom nucleotide analogs, and, as applicable to the embodiment beingdescribed, single (sense or antisense) and double-strandedpolynucleotides. Deoxyribonucleotides include deoxyadenosine,deoxycytidine, deoxyguanosine, and deoxythymidine. For purposes ofclarity, when referring herein to a nucleotide of a nucleic acid, whichcan be DNA or an RNA, the terms “adenosine”, “cytidine”, “guanosine”,and thymidine” are used. It is understood that if the nucleic acid isRNA, a nucleotide having a uracil base is uridine.

[0087] The term “nucleotide sequence complementary to the nucleotidesequence set forth in SEQ ID NO: x” refers to the nucleotide sequence ofthe complementary strand of a nucleic acid strand having SEQ ID NO: x.The term “complementary strand” is used herein interchangeably with theterm “complement”. The complement of a nucleic acid strand can be thecomplement of a coding strand or the complement of a non-coding strand.When referring to double stranded nucleic acids, the complement of anucleic acid having SEQ ID NO: x refers to the complementary strand ofthe strand having SEQ ID NO: x or to any nucleic acid having thenucleotide sequence of the complementary strand of SEQ ID NO: x. Whenreferring to a single stranded nucleic acid having the nucleotidesequence SEQ ID NO: x, the complement of this nucleic acid is a nucleicacid having a nucleotide sequence which is complementary to that of SEQID NO: x. The nucleotide sequences and complementary sequences thereofare always given in the 5′ to 3′ direction. The term “complement” and“reverse complement” are used interchangeably herein.

[0088] A “non-human animal” of the invention can include mammals such asrodents, non-human primates, sheep, goats, horses, dogs, cows, chickens,amphibians, reptiles, etc. Preferred non-human animals are selected fromthe rodent family including rat and mouse, most preferably mouse, thoughtransgenic amphibians, such as members of the Xenopus genus, andtransgenic chickens can also provide important tools for understandingand identifying agents which can affect, for example, embryogenesis andtissue formation. The term “chimeric animal” is used herein to refer toanimals in which an exogenous sequence is found, or in which anexogenous sequence is expressed in some but not all cells of the animal.The term “tissue-specific chimeric animal” indicates that an exogenoussequence is present and/or expressed or disrupted in some tissues, butnot others.

[0089] The term “operably linked” is intended to mean that the promoteris associated with the nucleic acid in such a manner as to facilitatetranscription of the nucleic acid from the promoter.

[0090] The term “polymorphism” refers to the coexistence of more thanone form of a gene or portion thereof. A portion of a gene of whichthere are at least two different forms, i.e., two different nucleotidesequences, is referred to as a “polymorphic region of a gene”. Apolymorphic region can be a single nucleotide, the identity of whichdiffers in different alleles. A polymorphic region can also be severalnucleotides long.

[0091] A “polymorphic gene” refers to a gene having at least onepolymorphic region.

[0092] The terms “protein”, “polypeptide” and “peptide” are usedinterchangeably herein when referring to a gene product.

[0093] The term “recombinant protein” refers to a polypeptide which isproduced by recombinant DNA techniques, wherein generally, DNA encodingthe polypeptide is inserted into a suitable expression vector which isin turn used to transform a host cell to produce the heterologousprotein.

[0094] A “regulatory element”, also termed herein “regulatory sequenceis intended to include elements which are capable of modulatingtranscription from a basic promoter and include elements such asenhancers and silencers. The term “enhancer”, also referred to herein as“enhancer element”, is intended to include regulatory elements capableof increasing, stimulating, or enhancing transcription from a basicpromoter. The termn “silencer”, also referred to herein as “silencerelement” is intended to include regulatory elements capable ofdecreasing, inhibiting, or repressing transcription from a basicpromoter. Regulatory elements are typically present in 5′ flankingregions of genes. However, regulatory elements have also been shown tobe present in other regions of a gene, in particular in introns. Thus,it is possible that SR-BI genes have regulatory elements located inintrons, exons, coding regions, and 3′ flanking sequences. Suchregulatory elements are also intended to be encompassed by the presentinvention and can be identified by any of the assays that can be used toidentify regulatory elements in 5′ flanking regions of genes.

[0095] The term “regulatory element” further encompasses “tissuespecific” regulatory elements, i.e., regulatory elements which effectexpression of the selected DNA sequence preferentially in specific cells(e.g., cells of a specific tissue). Gene expression occurspreferentially in a specific cell if expression in this cell type issignificantly higher than expression in other cell types. The term“regulatory element” also encompasses non-tissue specific regulatoryelements, i.e., regulatory elements which are active in most cell types.Furthermore, a regulatory element can be a constitutive regulatoryelement, i.e., a regulatory element which constitutively regulatestranscription, as opposed to a regulatory element which is inducible,i.e., a regulatory element which is active primarily in response to astimulus. A stimulus can be, e.g., a molecule, such as a hormone,cytokine, heavy metal, phorbol ester, cyclic AMP (cAMP), or retinoicacid.

[0096] Regulatory elements are typically bound by proteins, e.g.,transcription factors. The term “transcription factor” is intended toinclude proteins or modified forms thereof, which interactpreferentially with specific nucleic acid sequences, i.e., regulatoryelements, and which in appropriate conditions stimulate or represstranscription. Some transcription factors are active when they are inthe form of a monomer. Alternatively, other transcription factors areactive in the form of a dimer consisting of two identical proteins ordifferent proteins (heterodimer). Modified forms of transcriptionfactors are intended to refer to transcription factors having apostranslational modification, such as the attachment of a phosphategroup. The activity of a transcription factor is frequently modulated bya postranslational modification. For example, certain transcriptionfactors are active only if they are phosphorylated on specific residues.Alternatively, transcription factors can be active in the absence ofphosphorylated residues and become inactivated by phosphorylation. Alist of known transcription factors and their DNA binding site can befound, e.g., in public databases, e.g., TFMATRIX Transcription FactorBinding Site Profile database.

[0097] As used herein, the term “specifically hybridizes” or“specifically detects” refers to the ability of a nucleic acid moleculeof the invention to hybridize to at least approximately 6, 12, 20, 30,40, 50, 60, 70, 80, 90, 100, 110, 120, 130 or 140 consecutivenucleotides of either strand of an SR-BI gene.

[0098] “SR-BI” or “SR-BI receptor” refers to a class B scavengerreceptor that has been shown to bind HDL cholesterol and mediate uptakeinto cells (Acton, S. et al., Science 271:518-520). SR-BI has also beenshown to bind with high affinity to modified proteins (e.g acetylatedLDL, oxidized LDL, maleylated bovine serum albumin) and native LDL(Acton, et al., (1994) J Biochem. 269:21003-21009). Further, SR-BI hasbeen shown to bind anionic phospholipids, such as phosphatidylserine andphosphatidylinositol, but not zwitterionic phospholipids, such asphosphatidylcholine, phosphatidylethanolamine and sphingomyelin.Competition studies suggest that anionic phospholipids bind to SR-BI ata site close to or identical with the sites of native and modified LDLbinding and that the interaction may involve polyvalent binding viamultiple anionic phospholipid molecules (Rigotti, A.., S. Acton and M.Krieger (1995) J. Biochem 270:16221-16224). SR-BI has also been shown tobind to negatively charged liposomes and apoptotic cells. The humanSR-BI protein is described in Calvo et al. (1993) J. Biol. Chem.268:18929 and hamster SR-BI is described in International PatentApplication No. WO 96/00288 entitled “Class B1 and C1 ScavengerReceptors” by Acton, S. et al.

[0099] The term “SR-BI therapeutic” refers to various forms of SR-BIpolypeptides, as well as peptidomimetics, nucleic acids, or smallmolecules, which can modulate at least one activity of an SR-BI bymimicking or potentiating (agonizing) or inhibiting (antagonizing) theeffects of a naturally-occurring SR-BI polypeptide. An SR-BI therapeuticwhich mimics or potentiates the activity of a wild-type SR-BIpolypeptide is a “SR-BI agonist”. Conversely, an SR-BI therapeutic whichinhibits the activity of a wild-type SR-BI polypeptide is a “SR-BIantagonist”. SR-BI therapeutics can be used to treat diseases which areassociated with a specific SR-BI allele which encodes a protein havingan amino acid sequence that differs from that of the wild-type SR-BIprotein.

[0100] As used herein, the term “transfection” means the introduction ofa nucleic acid, e.g., an expression vector, into a recipient cell bynucleic acid-mediated gene transfer. The term “transduction” isgenerally used herein when the transfection with a nucleic acid is byviral delivery of the nucleic acid. “Transformation”, as used herein,refers to a process in which a cell's genotype is changed as a result ofthe cellular uptake of exogenous DNA or RNA, and, for example, thetransformed cell expresses a recombinant form of a polypeptide or, inthe case of anti-sense expression from the transferred gene, theexpression of a naturally-occurring form of the recombinant protein isdisrupted.

[0101] As used herein, the term “transgene” refers to a nucleic acidsequence which has been introduced into a cell. Daughter cells derivingfrom a cell in which a transgene has been introduced are also said tocontain the transgene (unless it has been deleted). A transgene canencode, e.g., a polypeptide, or an antisense transcript, partly orentirely heterologous, i.e., foreign, to the transgenic animal or cellinto which it is introduced, or, is homologous to an endogenous gene ofthe transgenic animal or cell into which it is introduced, but which isdesigned to be inserted, or is inserted, into the animal's genome insuch a way as to alter the genome of the cell into which it is inserted(e.g., it is inserted at a location which differs from that of thenatural gene or its insertion results in a knockout). Alternatively, atransgene can also be present in an episome. A transgene can include oneor more transcriptional regulatory sequence and any other nucleic acid,(e.g. intron), that may be necessary for optimal expression of aselected nucleic acid.

[0102] A “transgenic animal” refers to any animal, preferably anon-human animal, e.g. a mammal, bird or an amphibian, in which one ormore of the cells of the animal contain heterologous nucleic acidintroduced by way of human intervention, such as by transgenictechniques well known in the art. The nucleic acid is introduced intothe cell, directly or indirectly by introduction into a precursor of thecell, by way of deliberate genetic manipulation, such as bymicroinjection or by infection with a recombinant virus. The termgenetic manipulation does not include classical cross-breeding, or invitro fertilization, but rather is directed to the introduction of arecombinant DNA molecule. This molecule may be integrated within achromosome, or it may be extrachromosomally replicating DNA. In thetypical transgenic animals described herein, the transgene causes cellsto express a recombinant form of one of a protein, e.g. either agonisticor antagonistic forms. However, transgenic animals in which therecombinant gene is silent are also contemplated, as for example, theFLP or CRE recombinase dependent constructs described below. Moreover,“transgenic animal” also includes those recombinant animals in whichgene disruption of one or more genes is caused by human intervention,including both recombination and antisense techniques.

[0103] The term “treatment”, or “treating”, as used herein, is definedas the application or administration of a therapeutic agent to apatient, or application or administration of a therapeutic agent to anisolated tissue or cell line from a patient, who has a disease, asymptom of disease or a predisposition toward a disease, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve or affect the disease, the symptoms of disease or thepredisposition toward disease. A therapeutic agent includes, but is notlimited to, small molecules, peptides, antibodies, ribozymes andantisense oligonucleotides.

[0104] As used herein, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of preferred vector is an episome, i.e., a nucleicacid capable of extra-chromosomal replication. Preferred vectors arethose capable of autonomous replication and/or expression of nucleicacids to which they are linked. Vectors capable of directing theexpression of genes to which they are operatively linked are referred toherein as “expression vectors”. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of“plasmids” which refer generally to circular double stranded DNA loopswhich, in their vector form are not bound to the chromosome. In thepresent specification, “plasmid” and “vector” are used interchangeablyas the plasmid is the most commonly used form of vector. However, theinvention is intended to include such other forms of expression vectorswhich serve equivalent functions and which become known in the artsubsequently hereto.

[0105] The term “wild-type allele” refers to an allele of a gene which,when present in two copies in a subject results in a wild-typephenotype. There can be several different wild-type alleles of aspecific gene, since certain nucleotide changes in a gene may not affectthe phenotype of a subject having two copies of the gene with thenucleotide changes.

[0106] Nucleic Acids of the Present Invention

[0107] As described below, one aspect of the invention pertains toisolated nucleic acids comprising an intronic sequence of an SR-BI gene.In a preferred embodiment, the invention provides an intronic sequenceof the genomic DNA sequence encoding an SR-BI protein, comprising anintronic sequence shown in FIG. 2A-G or set forth in any of SEQ ID NOs.1-121 or complements thereof or homologues thereof. Other preferrednucleic acids of the invention include specific SR-BI alleles, whichdiffer from the allelic variant having the nucleotide sequence set forthin SEQ ID NO: 1 or SEQ ID NO: 3, or at least a portion thereof having apolymorphic region. Nucleic acids of the invention can function asprobes or primers, e.g., in methods for determining the identity of anallelic variant of an SR-BI polymorphic region. The nucleic acids of theinvention can also be used to determine whether a subject is at risk ofdeveloping a disease associated with a specific allelic variant of anSR-BI polymorphic region, e.g, a disease or disorder associated with anaberrant SR-BI activity. The nucleic acids of the invention can furtherbe used to prepare SR-BI polypeptides encoded by specific alleles, suchas mutant alleles. Such polypeptides can be used in gene therapy.Polypeptides encoded by specific SR-BI alleles, such as mutant SR-BIpolypeptides, can also be used for preparing reagents, e.g., antibodies,for detecting SR-BI proteins encoded by these alleles. Accordingly, suchreagents can be used to detect mutant SR-BI proteins.

[0108] Certain nucleic acids of the invention comprise an intronicsequence of an SR-BI gene. The term “SR-BI intronic sequence” refers toa nucleotide sequence of an intron of an SR-BI gene. An intronicsequence can be directly adjacent to an exon or located further awayfrom the exons. Preferred nucleic acids of the invention include anintronic sequence of an SR-BI gene which is adjacent to an exon andcomprises at least about 3 consecutive nucleotides, at least about 6consecutive nucleotides, at least about 9 consecutive nucleotides, atleast about 12 consecutive nucleotides, at least about 15 consecutivenucleotides, at least about 18 consecutive nucleotides, or at leastabout 20 consecutive nucleotides. Isolated nucleic acids which comprisean SR-BI intronic sequence which is immediately adjacent to an exon andcomprises at least about 25 consecutive nucleotides, at least about 30consecutive nucleotides, at least about 35 consecutive nucleotides, atleast about 40 consecutive nucleotides, at least about 50 consecutivenucleotides, or at least about 100 consecutive nucleotides are alsowithin the scope of the invention. Preferred isolated nucleic acids ofthe invention also include those having an SR-BI intronic sequencehaving a nucleotide sequence of at least about 10 nucleotides, at leastabout 15 nucleotides, at least about 20 nucleotides, at least about 25nucleotides, at least about 30 nucleotides, at least about 35nucleotides, at least about 40 nucleotides, at least about 50nucleotides or at least about 100 nucleotides. Other preferred nucleicacids of the invention can comprise an SR-BI intronic sequence havingless than about 10 nucleotides, provided that the nucleotide sequence isnovel. Yet other preferred isolated nucleic acids of the inventioninclude SR-BI intronic nucleic acid sequences of an SR-BI intron, havingat least about 150 consecutive nucleotides, at least about 200consecutive nucleotides, at least about 250 consecutive nucleotides, atleast about 300 consecutive nucleotides, at least about 350 consecutivenucleotides, at least about 400 consecutive nucleotides, at least about500 consecutive nucleotides or at least about 1000 consecutivenucleotides

[0109] Preferred nucleic acids of the invention comprise an SR-BIintronic sequence having a nucleotide sequence shown in FIG. 2A-G,and/or in any of SEQ ID Nos. 1-121, complement thereof, reversecomplement thereof or homologue thereof. In a preferred embodiment, theinvention provides an isolated nucleic acid comprising an SR-BI intronicwhich is at least about 70% 75%, 80%, 85%, 90%, 95%, or preferably atleast about 98%, and most preferably at least about 99% identical to anintronic nucleotide sequence shown in FIG. 2A-G or set forth in any ofSEQ ID NOS. 1-121 or a complement thereof. In fact, as described herein,several alleles of human SR-BI genes have been identified. The inventionis intended to encompass all of these alleles and SR-BI alleles not yetidentified, which can be identified, e.g, according to the methodsdescribed herein.

[0110] The invention also provides isolated nucleic acids comprising atleast one polymorphic region of an SR-BI gene having a nucleotidesequence which differs from the nucleotide sequence set forth in SEQ IDNO: 1 or SEQ ID NO: 3. Preferred nucleic acids have a polymorphic regionlocated in an exon of an SR-BI gene, such as exons 1, 3 or 8.Accordingly, preferred nucleic acids of the invention comprise anadenine at position 146 of exon 1 (as set forth in SEQ ID NO: 95), anadenine at position 119 of exon 3 (as set forth in SEQ ID NO: 96, and/ora thymidine at position 41 of exon 8 (as set forth in SEQ ID NO: 65).Preferred nucleic acids can also have a polymorphic region in an intron,e.g., intron 5 or 10. For example, the invention provides nucleic acidshaving a polymorphic nucleotide at position 54 of intron 5 and/or atposition −41 of intron 10. In a preferred embodiment, the nucleic acidhas a thymidine at position 54 of intron 5 (as set forth in SEQ ID NO:66) and/or a guanine at position −41 of intron 10 (as set forth in SEQID NO: 97). The nucleic acids can be genomic DNA, cDNA, or RNA (in whichcase, the nucleic acid has a uridine at position 54 of intron 5).

[0111] Also within the scope of the invention are isolated nucleic acidswhich encode an SR-BI protein, such as an SR-BI protein having an aminoacid sequence which differs from the amino acid sequence set forth inSEQ ID NOs. 2 and 4. Preferred nucleic acids encode an SR-BI polypeptidecomprising an amino acid sequence from SEQ ID NO: 2 or 4 in whichresidue 2 is a serine and/or in which residue 135 is an isoleucine.

[0112] Preferred nucleic acids of the invention are from vertebrategenes encoding SR-BI proteins. Particularly preferred vertebrate nucleicacids are mammalian nucleic acids. A particularly preferred nucleic acidof the invention is a human nucleic acid, such as a nucleic acidcomprising an SR-BI intronic sequence shown in FIG. 2A-G or set forth inany of SEQ ID NOS. 1-121 or complement thereof or an allele comprising anucleotide sequence set forth in SEQ ID NO: 65 or SEQ ID NO: 97.

[0113] Another aspect of the invention provides a nucleic acid whichhybridizes under appropriate stringency to an SR-BI intronic sequencehaving a nucleotide sequence shown in introns shown in FIG. 2A-G or inintronic sequences set forth in any of SEQ ID Nos. 1-121 or complementthereof. Appropriate stringency conditions which promote DNAhybridization, for example, 6.0 x sodium chloride/sodium citrate (SSC)at about 45° C., followed by a wash of 2.0×SSC at 50° C., are known tothose skilled in the art or can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Forexample, the salt concentration in the wash step can be selected from alow stringency of about 2.0×SSC at 50° C. to a high stringency of about0.2×SSC at 50° C. In addition, the temperature in the wash step can beincreased from low stringency conditions at room temperature, about 22°C., to high stringency conditions at about 65° C. Both temperature andsalt may be varied, or temperature or salt concentration may be heldconstant while the other variable is changed. In a preferred embodiment,a nucleic acid of the present invention will bind to at least about 20,preferably at least about 25, more preferably at least about 30 and mostpreferably at least about 50 consecutive nucleotides of a sequence shownin FIG. 2A-G or set forth in any of SEQ ID Nos. 1-121 under moderatelystringent conditions, for example at about 2.0×SSC and about 40° C. Evenmore preferred nucleic acids of the invention are capable of hybridizingunder stringent conditions to an intronic sequence of at least about 20,30, 40, or at least about 50 nucleotides as shown in FIG. 2A-G or as setforth in an intronic sequence of any of SEQ ID Nos. 1-121.

[0114] Hybridization, as described above, can be used to isolate nucleicacids comprising an SR-BI intron or portion thereof from various animalspecies. A comparison of these nucleic acids should be indicative ofintronic sequences which may have a regulatory or other function, sincethese regions are expected to be conserved among various species.Hybridization can also be used to isolate SR-BI alleles.

[0115] The nucleic acid of the invention can be single stranded DNA(e.g., an oligonucleotide), double stranded DNA (e.g., double strandedoligonucleotide) or RNA. Preferred nucleic acids of the invention can beused as probes or primers. Primers of the invention refer to nucleicacids which hybridize to a nucleic acid sequence which is adjacent tothe region of interest or which covers the region of interest and isextended. A primer can be used alone in a detection method, or a primercan be used together with at least one other primer or probe in adetection method. Primers can also be used to amplify at least a portionof a nucleic acid. Probes of the invention refer to nucleic acids whichhybridize to the region of interest and which are not further extended.For example, a probe is a nucleic acid which hybridizes to a polymorphicregion of an SR-BI gene, and which by hybridization or absence ofhybridization to the DNA of a subject will be indicative of the identityof the allelic variant of the polymorphic region of the SR-BI gene.

[0116] Numerous procedures for determining the nucleotide sequence of anucleic acid, or for determining the presence of mutations in nucleicacids include a nucleic acid amplification step, which can be carriedout by, e.g., polymerase chain reaction (PCR). Accordingly, in oneembodiment, the invention provides primers for amplifying portions of anSR-BI gene, such as portions of exons and/or portions of introns. In apreferred embodiment, the exons and/or sequences adjacent to the exonsof the human SR-BI gene will be amplified to, e.g., detect which allelicvariant of a polymorphic region is present in the SR-BI gene of asubject. Preferred primers comprise a nucleotide sequence complementaryto an SR-BI intronic sequence or a specific allelic variant of an SR-BIpolymorphic region and of sufficient length to selectively hybridizewith an SR-BI gene. In a preferred embodiment, the primer, e.g., asubstantially purified oligonucleotide, comprises a region having anucleotide sequence which hybridizes under stringent conditions to about6, 8, 10, or 12, preferably 25, 30, 40, 50, or 75 consecutivenucleotides of an SR-BI gene. In an even more preferred embodiment, theprimer is capable of hybridizing to an SR-BI intron and has a nucleotidesequence of an intronic sequence shown in FIG. 2A-G or set forth in anyof SEQ ID Nos. 1-121, complements thereof, allelic variants thereof, orcomplements of allelic variants thereof. For example, primers comprisinga nucleotide sequence of at least about 15 consecutive nucleotides, atleast about 20 nucleotides or having from about 15 to about 25nucleotides shown in FIG. 2A-G or set forth in any of SEQ ID NOS. 1-121or complement thereof are provided by the invention. Primers having asequence of more than about 25 nucleotides are also within the scope ofthe invention. Preferred primers of the invention are primers that canbe used in PCR for amplifying each of the exons of an SR-BI gene. Evenmore preferred primers of the invention have the nucleotide sequence setforth in any of SEQ ID Nos. 41-64 and 89-94 (see Table VII and X in theExamples).

[0117] Primers can be complementary to nucleotide sequences locatedclose to each other or further apart, depending on the use of theamplified DNA. For example, primers can be chosen such that they amplifyDNA fragments of at least about 10 nucleotides or as much as severalkilobases. Preferably, the primers of the invention will hybridizeselectively to nucleotide sequences located about 150 to about 350nucleotides apart.

[0118] For amplifying at least a portion of a nucleic acid, a forwardprimer (i.e., 5′ primer) and a reverse primer (i.e., 3′ primer) willpreferably be used. Forward and reverse primers hybridize tocomplementary strands of a double stranded nucleic acid, such that uponextension from each primer, a double stranded nucleic acid is amplified.A forward primer can be a primer having a nucleotide sequence or aportion of the nucleotide sequence shown in FIG. 2A-G or in SEQ ID Nos.1-40, 65, 66, and 95-97. A reverse primer can be a primer having anucleotide sequence or a portion of the nucleotide sequence that iscomplementary to a nucleotide sequence shown in FIG. 2A-G or in SEQ IDNos. 1-40, 65, 66, and 95-97. Preferred forward primers comprise anucleotide sequence set forth in SEQ ID Nos. 41, 43, 45, 47, 49, 51, 53,55, 57, 59, 61, 63, and 85 (shown in Table VII). Preferred reverseprimers comprise a nucleotide sequence set forth in SEQ ID Nos. 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, and 86. Preferred pairs ofprimers for amplifying each of the exons of human SR-BI are set forth inTable VII.

[0119] Yet other preferred primers of the invention are nucleic acidswhich are capable of selectively hybridizing to an allelic variant of apolymorphic region of an SR-BI gene. Thus, such primers can be specificfor an SR-BI gene sequence, so long as they have a nucleotide sequencewhich is capable of hybridizing to an SR-BI gene. Preferred primers arecapable of specifically hybridizing to an allelic variant in whichnucleotide 146 of exon 1 of human SR-BI is an adenine, e.g., a nucleicacid having SEQ ID NO: 95; an allelic variant in which nucleotide 119 ofexon 3 is an adenine, e.g., a nucleic acid having SEQ ID NO: 96; or anallelic variant in which nucleotide 41 of exon 8 of human SR-BI is athymidine, e.g., a nucleic acid having SEQ ID NO: 65. Other preferredprimers are capable of specifically hybridizing to an allelic variant inwhich nucleotide 54 of intron 5 is a thymidine, e.g., a nucleic acidhaving SEQ ID NO: 66 or nucleotide −41 of intron 10 is a guanine, e.g.,a nucleic acid having SEQ ID NO: 97. Such primers can be used, e.g., insequence specific oligonucleotide priming as described further herein.

[0120] The SR-BI nucleic acids of the invention can also be used asprobes, e.g., in therapeutic and diagnostic assays. For instance, thepresent invention provides a probe comprising a substantially purifiedoligonucleotide, which oligonucleotide comprises a region having anucleotide sequence that hybridizes under stringent conditions to atleast approximately 6, 8, 10 or 12, preferably about 25, 30, 40, 50 or75 consecutive nucleotides of an SR-BI gene. In one embodiment, theprobes preferably hybridize to an intron of an SR-BI gene, having anintronic nucleotide sequence shown in FIG. 2A-G or set forth in any ofSEQ ID Nos. 1-121, allelic variants thereof, complements thereof orcomplements of allelic variants thereof. In another embodiment, theprobes are capable of hybridizing to a nucleotide sequence encompassingan intron/exon border of an SR-BI gene.

[0121] Other preferred probes of the invention are capable ofhybridizing specifically to a region of an SR-BI gene which ispolymorphic. In an even more preferred embodiment of the invention, theprobes are capable of hybridizing specifically to one allelic variant ofan SR-BI gene having a nucleotide sequence which differs from thenucleotide sequence set forth in SEQ ID NO: 1 or 3. Such probes can thenbe used to specifically detect which allelic variant of a polymorphicregion of an SR-BI gene is present in a subject. The polymorphic regioncan be located in the promoter, exon, or intron sequences of an SR-BIgene.

[0122] For example, preferred probes of the invention are capable ofhybridizing specifically to a region overlapping nucleotide 146 of exon1 of the human SR-BI gene. In one embodiment, the probe overlappingnucleotide 146 of exon 1 is capable of hybridizing specifically to anucleotide sequence wherein nucleotide 146 is an adenine (as shown inSEQ ID NO: 95). Examples of such probes include a probe having thenucleotide sequence 5′ GCGGAGCAGCTCATGTCTGCG 3′ (SEQ ID NO: 98); 5′CTTTCGCGGAGCAGCTCATGTCTGCGCGCCT 3′ (SEQ ID NO: 99); and probes havingthe complement of these nucleotide sequences, i.e., 5′CGCAGACATGAGCTGCTCCGC 3′ (SEQ ID NO: 100); 5′AGGCGCGCAGACATGAGCTGCTCCGCCAAAG 3′ (SEQ ID NO: 101). The boldnucleotides represents the location of the nucleotide polymorphism. Inanother embodiment, the probe overlapping nucleotide 146 of exon 1 iscapable of specifically hybridizing to a nucleotide sequence whereinnucleotide 146 is a guanine (as shown in FIG. 2A-G and set forth in SEQID NO: 5). Examples of such probes include a probe having the nucleotidesequence 5′ GCGGAGCAGCGCATGTCTGCG 3′ (SEQ ID NO: 102);CTTTCGCGGAGCAGCGCATGTCTGCGCGCCT 3′ (SEQ ID NO: 103) and probes havingthe complement of these nucleotide sequences, i.e., 5′CGCAGACATGCGCTGCTCCGC 3′ (SEQ ID NO: 104); 5′AGGCGCGCAGACATGCGCTGCTCCGCCAAAG 3′ (SEQ ID NO: 105).

[0123] Preferred probes of the invention are capable of hybridizingspecifically to a region overlapping nucleotide 119 of exon 3 of thehuman SR-BI gene. In one embodiment, the probe overlapping nucleotide119 of exon 3 is capable of hybridizing specifically to a nucleotidesequence wherein nucleotide 119 is an adenine (as shown in SEQ ID NO:96). Examples of such probes include a probe having the nucleotidesequence 5′ TTGGGCATGATGATGTAGACG 3′ (SEQ ID NO: 106); 5′GGATGTTGGGCATGATGATGTAGACGCTCTC 3′ (SEQ ID NO: 107); and probes havingthe complement of these nucleotide sequences, i.e., 5′CGACTACATCATCATGCCCAA 3′ (SEQ ID NO: 108); 5′GAGAGCGACTACATCATCATGCCCAACATCC 3′ (SEQ ID NO: 109). The boldnucleotides represents the location of the nucleotide polymorphism. Inanother embodiment, the probe overlapping nucleotide 119 of exon 3 iscapable of specifically hybridizing to a nucleotide sequence whereinnucleotide 119 is a guanine (as shown in FIG. 2A-G and set forth in SEQID NO: 7). Examples of such probes include a probe having the nucleotidesequence 5′ TTGGGCATGAGGATGTAGACG 3′ (SEQ ID NO: 110);GGATGTTGGGCATGAGGATGTAGACGCTCTC 3′ (SEQ ID NO: 111) and probes havingthe complement of these nucleotide sequences, i.e., 5′CGACTACATCCTCATGCCCAA 3′ (SEQ ID NO: 112); 5′GAGAGCGACTACATCCATCATGCCCAACATCC 3′ (SEQ ID NO: 113).

[0124] Other preferred probes of the invention are capable ofhybridizing specifically to a region overlapping nucleotide 41 of exon 8of the human SR-BI gene. In one embodiment, the probe overlappingnucleotide 41 of exon 8 is capable of hybridizing specifically to anucleotide sequence wherein nucleotide 41 is a thymidine (as shown inSEQ ID NO: 65). Examples of such probes include a probe having thenucleotide sequence 5′ AACCGGGTCAGCGTTGAGGA 3′ (SEQ ID NO: 67); 5′TGCCAGAACCGGGTCAGCGTTGAGGAAGTGA 3′ (SEQ ID NO: 68); and probes havingthe complement of these nucleotide sequences, i.e., 5′TCCTCAACGCTGACCCGGTT 3′ (SEQ ID NO: 69); 5′TCACTTCCTCAACGCTGACCCGGTTCTGGCA 3′ (SEQ ID NO: 70). The bold nucleotidesrepresents the location of the nucleotide polymorphism. In anotherembodiment, the probe overlapping nucleotide 41 of exon 8 is capable ofspecifically hybridizing to a nucleotide sequence wherein nucleotide 41is a cytidine (as shown in FIG. 2A-G and set forth in SEQ ID NO: 12).Examples of such probes include a probe having the nucleotide sequence5′ AACCGGGTCGGCGTTGATGA 3′ (SEQ ID NO: 71);TGCCAGAACCGGGTCGGCGTTGATGAAGTGA 3′ (SEQ ID NO: 72) and probes having thecomplement of these nucleotide sequences, i.e., 5′ TCATCAACGCCGACCCGGTT3′ (SEQ ID NO: 73); 5′ TCACTTCATCAACGCCGACCCGGTTCTGGCA 3′ (SEQ ID NO:74).

[0125] Yet other preferred probes of the invention are capable ofhybridizing specifically to a region overlapping nucleotide 54 of intron5 of the human SR-BI gene. In one embodiment, the probe overlappingnucleotide 54 of intron 5 is capable of hybridizing specifically to anucleotide sequence wherein nucleotide 54 is a cytidine (as shown inFIG. 2A-G and set forth in SEQ ID NOS. 9 and 26). Examples of suchprobes include a probe having the nucleotide sequence 5′AGCCATGGCCGGGCCCACCCT 3′ (SEQ ID NO: 75); 5′CGAGCAGCCATGGCCGGGCCCACCCTCCCCT 3′ (SEQ ID NO: 76); and probes havingthe complement of these nucleotide sequences, i.e., 5′AGGGTGGGCCCGGCCATGGCT 3′ (SEQ ID NO: 77); 5′AGGGGAGGGTGGGCCCGGCCATGGCTGCTCG 3′ (SEQ ID NO: 78). In anotherembodiment, the probe overlapping nucleotide 54 of intron 5 is capableof specifically hybridizing to a nucleotide sequence wherein nucleotide54 is a thymidine (as shown in SEQ ID NO: 66). Examples of such probesinclude a probe having the nucleotide sequence 5′ AGCCATGGCCAGGCCCACCCT3′ (SEQ ID NO: 79); 5′ CGAGCAGCCATGGCCAGGCCCACCCTCCCCT 3′ (SEQ ID NO:80); and probes having the complement of these nucleotide sequences,i.e., 5′ AGGGTGGGCCTGGCCATGGCT 3′ (SEQ ID NO: 81); 5′AGGGGAGGGTGGGCCTGGCCATGGCTGCTCG 3′ (SEQ ID NO: 82).

[0126] Still other preferred probes of the invention are capable ofhybridizing specifically to a region overlapping nucleotide −41 ofintron 10 of the human SR-BI gene. In one embodiment, the probeoverlapping nucleotide −41 of intron 10 is capable of hybridizingspecifically to a nucleotide sequence wherein nucleotide −41 is aguanine (as shown in SEQ ID NO: 97). Examples of such probes include aprobe having the nucleotide sequence 5′ TGGGGCCGCACGCTGCGGGCT 3′ (SEQ IDNO: 114); 5′ TGAGCTGGGGCCGCACGCTGCGGGCTACAGC 3′ (SEQ ID NO: 115); andprobes having the complement of these nucleotide sequences, i.e., 5′AGCCCGCAGCGTGCGGCCCCA 3′ (SEQ ID NO: 116); 5′GCTGTAGCCCGCAGCGTGCGGCCCCAGCTCA 3′ (SEQ ID NO: 117). The boldnucleotides represents the location of the nucleotide polymorphism. Inanother embodiment, the probe overlapping nucleotide −41 of intron 10 iscapable of specifically hybridizing to a nucleotide sequence whereinnucleotide −41 is a cytidine (as shown in FIG. 2A-G and set forth in SEQID NO: 15). Examples of such probes include a probe having thenucleotide sequence 5′ TGGGGCCGCAGGCTGCGGGCT 3′ (SEQ ID NO: 118);TGAGCTGGGGCCGCAGGCTGCGGGCTACAGC 3′ (SEQ ID NO: 119) and probes havingthe complement of these nucleotide sequences, i.e., 5′AGCCCGCAGCCTGCGGCCCCA 3′ (SEQ ID NO: 120); 5′GCTGTAGCCCGCAGCCTGCGGCCCCAGCTCA 3′ (SEQ ID NO: 121).

[0127] Particularly, preferred probes of the invention have a number ofnucleotides sufficient to allow specific hybridization to the targetnucleotide sequence. Where the target nucleotide sequence is present ina large fragment of DNA, such as a genomic DNA fragment of several tensor hundreds of kilobases, the size of the probe may have to be longer toprovide sufficiently specific hybridization, as compared to a probewhich is used to detect a target sequence which is present in a shorterfragment of DNA. For example, in some diagnostic methods, a portion ofan SR-BI gene may first be amplified and thus isolated from the rest ofthe chromosomal DNA and then hybridized to a probe. In such a situation,a shorter probe will likely provide sufficient specificity ofhybridization. For example, a probe having a nucleotide sequence ofabout 10 nucleotides may be sufficient.

[0128] In preferred embodiments, the probe or primer further comprises alabel attached thereto, which, e.g, is capable of being detected, e.g.the label group is selected from amongst radioisotopes, fluorescentcompounds, enzymes, and enzyme co-factors.

[0129] In a preferred embodiment of the invention, the isolated nucleicacid, which is used, e.g., as a probe or a primer, is modified, such asto become more stable. Exemplary nucleic acid molecules which aremodified include phosphoramidate, phosphothioate and methylphosphonateanalogs of DNA (see also U.S. Pat Nos. 5,176,996; 5,264,564; and5,256,775).

[0130] The nucleic acids of the invention can also be modified at thebase moiety, sugar moiety, or phosphate backbone, for example, toimprove stability of the molecule. The nucleic acids, e.g., probes orprimers, may include other appended groups such as peptides (e.g., fortargeting host cell receptors in vivo), or agents facilitating transportacross the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl.Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad.Sci. 84:648-652; PCT Publication No. W088/09810, published Dec. 15,1988), hybridization-triggered cleavage agents. (See, e.g., Krol et al.,1988, BioTechniques 6:958-976) or intercalating agents. (See, e.g., Zon,1988, Pharm. Res. 5:539-549). To this end, the nucleic acid of theinvention may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

[0131] The isolated nucleic acid comprising an SR-BI intronic sequencemay comprise at least one modified base moiety which is selected fromthe group including but not limited to 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytidine,5-(carboxyhydroxymethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytidine, 5-methylcytidine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytidine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxycetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

[0132] The isolated nucleic acid may also comprise at least one modifiedsugar moiety selected from the group including but not limited toarabinose, 2-fluoroarabinose, xylulose, and hexose.

[0133] In yet another embodiment, the nucleic acid comprises at leastone modified phosphate backbone selected from the group consisting of aphosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal or analog thereof.

[0134] In yet a further embodiment, the nucleic acid is an α-anomericoligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nuc. Acids Res. 15:6625-6641). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330).

[0135] Any nucleic acid fragment of the invention can be preparedaccording to methods well known in the art and described, e.g, inSambrook, J. Fritsch, E. F. , and Maniatis, T. (1989) Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. For example, discrete fragments of the DNA can be preparedand cloned using restriction enzymes. Alternatively, discrete fragmentscan be prepared using the Polymerase Chain Reaction (PCR) using primershaving an appropriate sequence.

[0136] Oligonucleotides of the invention may be synthesized by standardmethods known in the art, e.g by use of an automated DNA synthesizer(such as are commercially available from Biosearch, Applied Biosystems,etc.). As examples, phosphorothioate oligonucleotides may be synthesizedby the method of Stein et al. (1988, Nucl. Acids Res. 16:3209),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

[0137] The invention also provides vectors and plasmids containing thenucleic acids of the invention. For example, in one embodiment, theinvention provides a vector comprising at least a portion of an SR-BIgene comprising a polymorphic region and/or intronic sequence. Thus, theinvention provides vectors for expressing at least a portion of thenewly identified allelic variants of the human SR-BI gene, as well asother allelic variants, having a nucleotide sequence which is differentfrom the nucleotide sequence disclosed in Calvo and Vega, supra. Theallelic variants can be expressed in eukaryotic cells, e.g., cells of asubject, or in prokaryotic cells.

[0138] In one embodiment, the vector comprising at least a portion of anSR-BI allele is introduced into a host cell, such that a protein encodedby the allele is synthesized. The SR-BI protein produced can be used,e.g., for the production of antibodies, which can be used, e.g., inmethods for detecting mutant forms of SR-BI. Alternatively, the vectorcan be used for gene therapy, and be, e.g., introduced into a subject toproduce SR-BI protein. Host cells comprising a vector having at least aportion of an SR-BI gene are also within the scope of the invention.

[0139] Polypeptides of the present invention

[0140] The present invention makes available isolated SR-BIpolypeptides, such as SR-BI polypeptides which are encoded by specificallelic variants of SR-BI, such as those identified herein. Accordingly,preferred SR-BI polypeptides of the invention have an amino acidsequence which differs from SEQ ID NOs. 2 and 4. In one embodiment, theSR-BI polypeptides are isolated from, or otherwise substantially free ofother cellular proteins. The term “substantially free of other cellularproteins” (also referred to herein as “contaminating proteins”) or“substantially pure or purified preparations” are defined asencompassing preparations of SR-BI polypeptides having less than about20% (by dry weight) contaminating protein, and preferably having lessthan about 5% contaminating protein. Functional forms of the subjectpolypeptides can be prepared, for the first time, as purifiedpreparations by using a cloned gene as described herein.

[0141] Preferred SR-BI proteins of the invention have an amino acidsequence which is at least about 60%, 70%, 80%, 85%, 90%, or 95%identical or homologous to an amino acid sequence of SEQ ID NOs. 2 or 4.Even more preferred SR-BI proteins comprise an amino acid sequence whichis at least about 97, 98, or 99% homologous or identical to an aminoacid sequence of SEQ ID NO. 2 or 4. Such proteins can be recombinantproteins, and can be, e.g., produced in vitro from nucleic acidscomprising a specific allele of an SR-BI polymorphic region. Forexample, recombinant polypeptides preferred by the present invention canbe encoded by a nucleic acid, which is at least 85% homologous and morepreferably 90% homologous and most preferably 95% homologous with anucleotide sequence set forth in SEQ ID NOS. 1 or 3, and comprises anallele of a polymorphic region that differs from that set forth in SEQID NOs. 1 and 3. Polypeptides which are encoded by a nucleic acid thatis at least about 98-99% homologous with the sequence of SEQ ID NOs: 1or 3 and comprises an allele of a polymorphic region that differs fromthat set forth in SEQ ID NOs. 1 and 3 are also within the scope of theinvention.

[0142] In a preferred embodiment, an SR-BI protein of the presentinvention is a mammalian SR-BI protein. In an even more preferredembodiment, the SR-BI protein is a human protein, such as an SR-BIpolypeptide comprising an amino acid sequence from SEQ ID NO. 2 in whichamino acid 2 is a serine and/or amino acid 135 is an isoleucine. Otherpreferred SR-BI polypeptides comprise an amino acid sequence from SEQ IDNO: 4 in which amino acid 2 is a serine.

[0143] SR-BI polypeptides preferably are capable of functioning in oneof either role of an agonist or antagonist of at least one biologicalactivity of a wild-type (“authentic”) SR-BI protein of the appendedsequence listing. The term “evolutionarily related to”, with respect toamino acid sequences of SR-BI proteins, refers to both polypeptideshaving amino acid sequences which have arisen naturally, and also tomutational variants of human SR-BI polypeptides which are derived, forexample, by combinatorial mutagenesis.

[0144] Full length proteins or fragments corresponding to one or moreparticular motifs and/or domains or to arbitrary sizes, for example, atleast 5, 10, 25, 50, 75 and 100, amino acids in length are within thescope of the present invention.

[0145] Isolated peptidyl portions of SR-BI proteins can be obtained byscreening peptides recombinantly produced from the correspondingfragment of the nucleic acid encoding such peptides. In addition,fragments can be chemically synthesized using techniques known in theart such as conventional Merrifield solid phase f-Moc or t-Bocchemistry. For example, an SR-BI polypeptide of the present inventionmay be arbitrarily divided into fragments of desired length with nooverlap of the fragments, or preferably divided into overlappingfragments of a desired length. The fragments can be produced(recombinantly or by chemical synthesis) and tested to identify thosepeptidyl fragments which can function as either agonists or antagonistsof a wild-type (e.g., “authentic”) SR-BI protein.

[0146] In general, polypeptides referred to herein as having an activity(e.g., are “bioactive”) of an SR-BI protein are defined as polypeptideswhich mimic or antagonize all or a portion of the biological/biochemicalactivities of an SR-BI protein having SEQ ID NOs 2 or 4, such as theability to bind lipids. Other biological activities of the subject SR-BIproteins are described herein or will be reasonably apparent to thoseskilled in the art. According to the present invention, a polypeptidehas biological activity if it is a specific agonist or antagonist of anaturally-occurring form of an SR-BI protein.

[0147] Assays for determining whether an SR-BI protein or variantthereof, has one or more biological activities are well known in theart.

[0148] Other preferred proteins of the invention are those encoded bythe nucleic acids set forth in the section pertaining to nucleic acidsof the invention. In particular, the invention provides fusion proteins,e.g., SR-BI-immunoglobulin fusion proteins. Such fusion proteins canprovide, e.g., enhanced stability and solubility of SR-BI proteins andmay thus be useful in therapy. Fusion proteins can also be used toproduce an immunogenic fragment of an SR-BI protein. For example, theVP6 capsid protein of rotavirus can be used as an immunologic carrierprotein for portions of the SR-BI polypeptide, either in the monomericform or in the form of a viral particle. The nucleic acid sequencescorresponding to the portion of a subject SR-BI protein to whichantibodies are to be raised can be incorporated into a fusion geneconstruct which includes coding sequences for a late vaccinia virusstructural protein to produce a set of recombinant viruses expressingfusion proteins comprising SR-BI epitopes as part of the virion. It hasbeen demonstrated with the use of immunogenic fusion proteins utilizingthe Hepatitis B surface antigen fusion proteins that recombinantHepatitis B virions can be utilized in this role as well. Similarly,chimeric constructs coding for fusion proteins containing a portion ofan SR-BI protein and the poliovirus capsid protein can be created toenhance immunogenicity of the set of polypeptide antigens (see, forexample, EP Publication No: 0259149; and Evans et al. (1989) Nature339:385; Huang et al. (1988) J. Virol. 62:3855; and Schlienger et al.(1992) J. Virol. 66:2).

[0149] The Multiple antigen peptide system for peptide-basedimmunization can also be utilized to generate an immunogen, wherein adesired portion of an SR-BI polypeptide is obtained directly fromorgano-chemical synthesis of the peptide onto an oligomeric branchinglysine core (see, for example, Posnett et al. (1988) JBC 263:1719 andNardelli et al. (1992) J. Immunol. 148:914). Antigenic determinants ofSR-BI proteins can also be expressed and presented by bacterial cells.

[0150] In addition to utilizing fusion proteins to enhanceimmunogenicity, it is widely appreciated that fusion proteins can alsofacilitate the expression of proteins, and accordingly, can be used inthe expression of the SR-BI polypeptides of the present invention. Forexample, SR-BI polypeptides can be generated asglutathione-S-transferase (GST-fusion) proteins. Such GST-fusionproteins can enable easy purification of the SR-BI polypeptide, as forexample by the use of glutathione-derivatized matrices (see, forexample, Current Protocols in Molecular Biology, eds. Ausubel et al.(N.Y.: John Wiley & Sons, 1991)).

[0151] The present invention further pertains to methods of producingthe subject SR-BI polypeptides. For example, a host cell transfectedwith a nucleic acid vector directing expression of a nucleotide sequenceencoding the subject polypeptides can be cultured under appropriateconditions to allow expression of the peptide to occur. Suitable mediafor cell culture are well known in the art. The recombinant SR-BIpolypeptide can be isolated from cell culture medium, host cells, orboth using techniques known in the art for purifying proteins includingion-exchange chromatography, gel filtration chromatography,ultrafiltration, electrophoresis, and immunoaffinity purification withantibodies specific for such peptide. In a preferred embodiment, therecombinant SR-BI polypeptide is a fusion protein containing a domainwhich facilitates its purification, such as GST fusion protein.

[0152] Moreover, it will be generally appreciated that, under certaincircumstances, it may be advantageous to provide homologues of one ofthe subject SR-BI polypeptides which function in a limited capacity asone of either an SR-BI agonist (mimetic) or an SR-BI antagonist, inorder to promote or inhibit only a subset of the biological activitiesof the naturally-occurring form of the protein. Thus, specificbiological effects can be elicited by treatment with a homologue oflimited function, and with fewer side effects relative to treatment withagonists or antagonists which are directed to all of the biologicalactivities of naturally occurring forms of SR-BI proteins.

[0153] Homologues of each of the subject SR-BI proteins can be generatedby mutagenesis, such as by discrete point mutation(s), or by truncation.For instance, mutation can give rise to homologues which retainsubstantially the same, or merely a subset, of the biological activityof the SR-BI polypeptide from which it was derived. Alternatively,antagonistic forms of the protein can be generated which are able toinhibit the function of the naturally occurring form of the protein,such as by competitively binding to an SR-BI receptor.

[0154] The recombinant SR-BI polypeptides of the present invention alsoinclude homologues of SR-BI polypeptides which differ from the SR-BIproteins having SEQ ID NO. 2 or 4, such as versions of those proteinwhich are resistant to proteolytic cleavage, as for example, due tomutations which alter ubiquitination or other enzymatic targetingassociated with the protein.

[0155] SR-BI polypeptides may also be chemically modified to createSR-BI derivatives by forming covalent or aggregate conjugates with otherchemical moieties, such as glycosyl groups, lipids, phosphate, acetylgroups and the like. Covalent derivatives of SR-BI proteins can beprepared by linking the chemical moieties to functional groups on aminoacid sidechains of the protein or at the N-terminus or at the C-terminusof the polypeptide.

[0156] Modification of the structure of the subject SR-BI polypeptidescan be for such purposes as enhancing therapeutic or prophylacticefficacy, stability (e.g., ex vivo shelf life and resistance toproteolytic degradation), or post-translational modifications (e.g., toalter phosphorylation pattern of protein). Such modified peptides, whendesigned to retain at least one activity of the naturally-occurring formof the protein, or to produce specific antagonists thereof, areconsidered functional equivalents of the SR-BI polypeptides described inmore detail herein. Such modified peptides can be produced, forinstance, by amino acid substitution, deletion, or addition. Thesubstitutional variant may be a substituted conserved amino acid or asubstituted non-conserved amino acid.

[0157] For example, it is reasonable to expect that an isolatedreplacement of a leucine with an isoleucine or valine, an aspartate witha glutamate, a threonine with a serine, or a similar replacement of anamino acid with a structurally related amino acid (i.e. isosteric and/orisoelectric mutations) will not have a major effect on the biologicalactivity of the resulting molecule. Conservative replacements are thosethat take place within a family of amino acids that are related in theirside chains. Genetically encoded amino acids can be divided into fourfamilies: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine,histidine; (3) nonpolar =alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine. In similarfashion, the amino acid repertoire can be grouped as (1)acidic=aspartate, glutamate; (2) basic=lysine, arginine histidine, (3)aliphatic=glycine, alanine, valine, leucine, isoleucine, serine,threonine, with serine and threonine optionally be grouped separately asaliphatic-hydroxyl; (4) aromatic=phenylalanine, tyrosine, tryptophan;(5) amide=asparagine, glutamine; and (6) sulfur -containing=cysteine andmethionine. (see, for example, Biochemistry, 2^(nd) ed., Ed. by L.Stryer, WH Freeman and Co.: 1981). Whether a change in the amino acidsequence of a peptide results in a functional SR-BI homologue (e.g.,functional in the sense that the resulting polypeptide mimics orantagonizes the wild-type form) can be readily determined by assessingthe ability of the variant peptide to produce a response in cells in afashion similar to the wild-type protein, or competitively inhibit sucha response. Polypeptides in which more than one replacement has takenplace can readily be tested in the same manner.

[0158] Kits

[0159] As set forth herein, the invention provides methods, e.g.,diagnostic and therapeutic methods, e.g., for determining the type ofallelic variant of a polymorphic region present in an SR-BI gene, suchas a human SR-BI gene (e.g., EX8 or IVS5). In preferred embodiments, themethods use probes or primers comprising nucleotide sequences which arecomplementary to an SR-BI intronic sequence or to a polymorphic regionof an SR-BI gene. Accordingly, the invention provides kits forperforming these methods.

[0160] In a preferred embodiment, the invention provides a kit fordetermining whether a subject has or is at risk of developing a diseaseor condition associated with a specific allelic variant of an SR-BIpolymorphic region. In another preferred embodiment, the inventionprovides a kit for predicting the response (e.g., lowering HDL levels)to HRT in a subject based on the presence or absence of an SR-BI variant(e.g. EX8 or IVS5). In an even more preferred embodiment, the disease ordisorder is characterized by an abnormal SR-BI activity. In an even morepreferred embodiment, the invention provides a kit for determiningwhether a subject has or is at risk of developing a cardiovasculardisease, e.g., ischemia, restenosis, congestive heart failure,atherosclerosis, aberrant lipid (e.g., cholesterol), lipoprotein (e.g.,HDL, LDL) or triglyceride levels, gallstone formation, diabetes, or anabnormal body mass index, e.g, obesity or cachexia. A preferred kitprovides reagents for determining whether a female subject is has or islikely to develop high LDL levels or a high BMI or whether a malesubject has or is likely to develop low HDL levels. Another preferredkit provides reagents for determining whether a subject (e.g., a male ora female subject) has or is likely to develop low HDL levels.

[0161] Preferred kits comprise at least one probe or primer which iscapable of specifically hybridizing to an SR-BI sequence or polymorphicregion and instructions for use. The kits preferably comprise at leastone of the above described nucleic acids, e.g., including nucleic acidshybridizing to an exon/intron border. Preferred kits for amplifying atleast a portion of an SR-BI gene, e.g., an exon, comprise two primers,at least one of which is capable of hybridizing to an SR-BI intronicsequence or an allelic variant sequence. Even more preferred kitscomprise a pair of primers selected from the group consisting of SEQ IDNO: 41 and SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44, SEQ ID NO: 45and SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48, SEQ ID NO: 49 andSEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52, SEQ ID NO: 53 and SEQ IDNO: 54, SEQ ID NO: 55 and SEQ ID NO: 56, SEQ ID NO: 57 and SEQ ID NO:58, SEQ ID NO: 59 and SEQ ID NO: 60, SEQ ID NO: 61 and SEQ ID NO: 62,SEQ ID NO: 63 and SEQ ID NO: 64, SEQ ID NO: 85 and SEQ ID NO: 86, SEQ IDNO: 89 and SEQ ID NO: 90, SEQ ID NO: 91 and SEQ ID NO: 92, and SEQ IDNO: 93 and SEQ ID NO: 94.

[0162] The kits of the invention can also comprise one or more controlnucleic acids or reference nucleic acids, such as nucleic acidscomprising an SR-BI intronic sequence. For example, a kit can compriseprimers for amplifying a polymorphic region of an SR-BI gene and acontrol DNA corresponding to such an amplified DNA and having thenucleotide sequence of a specific allelic variant. Thus, directcomparison can be performed between the DNA amplified from a subject andthe DNA having the nucleotide sequence of a specific allelic variant. Inone embodiment, the control nucleic acid comprises at least a portion ofan SR-BI gene of an individual, who does not have a cardiovasculardisease, aberrant lipid levels, gallstones, or a disease or disorderassociated with an aberrant SR-BI activity.

[0163] Yet other kits of the invention comprise at least one reagentnecessary to perform the assay. For example, the kit can comprise anenzyme. Alternatively the kit can comprise a buffer or any othernecessary reagent.

[0164] Predictive Medicine

[0165] The invention further features predictive medicines, which arebased, at least in part, on determination of the identity of SR-BIpolymorphic regions which are associated with specific diseases ordisorders.

[0166] For example, information obtained using the diagnostic assaysdescribed herein (alone or in conjunction with information on anothergenetic defect, which contributes to the same disease) is useful fordiagnosing or confirming that a symptomatic subject has an allele of apolymorphic region which is associated with a particular disease ordisorder. Alternatively, the information (alone or in conjunction withinformation on another genetic defect, which contributes to the samedisease) can be used prognostically for predicting whether anon-symptomatic subject is likely to develop a disease or condition,which is associated with one or more specific alleles of SR-BIpolymorphic regions in a subject. Based on the prognostic information, adoctor can recommend a regimen (e.g. diet or exercise) or therapeuticprotocol, useful for preventing or prolonging onset of the particulardisease or condition in the individual.

[0167] In addition, knowledge of the identity of a particular SR-BIallele in an individual (the SR-BI genetic profile), alone or inconjunction with information on other genetic defects contributing tothe same disease (the genetic profile of the particular disease) allowscustomization of therapy for a particular disease to the individual'sgenetic profile, the goal of “pharnacogenomics”. For example, anindividual's SR-BI genetic profile or the genetic profile of a diseaseor condition associated with a specific allele of an SR-BI polymorphicregion, can enable a doctor: 1) to more effectively prescribe a drugthat will address the molecular basis of the disease or condition; and2) to better determine the appropriate dosage of a particular drug. Forexample, the expression level of SR-BI proteins, alone or in conjunctionwith the expression level of other genes, known to contribute to thesame disease, can be measured in many patients at various stages of thedisease to generate a transcriptional or expression profile of thedisease. Expression patterns of individual patients can then be comparedto the expression profile of the disease to determine the appropriatedrug and dose to administer to the patient.

[0168] The ability to target populations expected to show the highestclinical benefit, based on the SR-BI or disease genetic profile, canenable: 1) the repositioning of marketed drugs with disappointing marketresults; 2) the rescue of drug candidates whose clinical development hasbeen discontinued as a result of safety or efficacy limitations, whichare patient subgroup-specific; and 3) an accelerated and less costlydevelopment for drug candidates and more optimal drug labeling (e.g.since the use of SR-BI as a marker is useful for optimizing effectivedose).

[0169] These and other methods are described in further detail in thefollowing sections.

[0170] Prognostic and Diagnostic Assays

[0171] The present methods provide means for determining if a subjecthas (diagnostic) or is at risk of developing (prognostic) a disease,condition or disorder that is associated a specific SR-BI allele, e.g.,a body mass disorder or an abnormal lipid level (HDL and LDL) anddisorders resulting therefrom.

[0172] The present invention provides methods for determining themolecular structure of an SR-BI gene, such as a human SR-BI gene, or aportion thereof. In one embodiment, determining the molecular structureof at least a portion of an SR-BI gene comprises determining theidentity of the allelic variant of at least one polymorphic region of anSR-BI gene. A polymorphic region of an SR-BI gene can be located in anexon, an intron, at an intron/exon border, or in the promoter of theSR-BI gene.

[0173] The invention provides methods for determining whether a subjecthas, or is at risk of developing, a disease or condition associated witha specific allelic variant of a polymorphic region of an SR-BI gene.Such diseases can be associated with an aberrant SR-BI activity, e.g.,abnormal binding to a lipid, or an aberrant SR-BI protein level. Anaberrant SR-BI protein level can result from an aberrant transcriptionor post transcriptional regulation. Thus, allelic differences inspecific regions of an SR-BI gene can result in differences of SR-BIprotein due to differences in regulation of expression. In particular,some of the identified polymorphisms in the human SR-BI gene may beassociated with differences in the level of transcription, RNAmaturation, splicing, or translation of the SR-BI gene or transcriptionproduct. This invention further provides methods for predicting theeffect of HRT on lipid level (e.g. HDL or LDL), in females, based on theidentification of specific allelic variants of an SR-B1 gene (e.g., EX8or IVS5).

[0174] Analysis of one or more SR-BI polymorphic region in a subject canbe useful for predicting whether a subject has or is likely to develop abody mass disorder, an abnormal lipoprotein or lipid level and disordersresulting therefrom, such as cardiovascular disorders, diabetes andgallstone formation, or for predicting the effect of HRT on lipid levelin a subject.

[0175] In addition, since SR-BI is a receptor that is capable of bindingto various lipid related molecules, it is likely that specific allelesof the SR-BI gene are associated with other diseases or conditionsinvolving an inappropriate lipid transfer or metabolism, e.g.,atherosclerosis or a biliary disorder, such as gallstone formation.Accordingly, the invention provides diagnostic and prognostic assays fordetermining whether a subject is at risk of developing a diseasecharacterized by an abnormal lipid level, e.g, atherosclerosis or gallstone formation.

[0176] In preferred embodiments, the methods of the invention can becharacterized as comprising detecting, in a sample of cells from thesubject, the presence or absence of a specific allelic variant of one ormore polymorphic regions of an SR-BI gene. The allelic differences canbe: (i) a difference in the identity of at least one nucleotide or (ii)a difference in the number of nucleotides, which difference can be asingle nucleotide or several nucleotides. The invention also providesmethods for detecting differences in SR-BI genes such as chromosomalrearrangements, e.g, chromosomal dislocation. The invention can also beused in prenatal diagnostics.

[0177] A preferred detection method is allele specific hybridizationusing probes overlapping the polymorphic site and having about 5, 10,20, 25, or 30 nucleotides around the polymorphic region. Examples ofprobes for detecting specific allelic variants of the polymorphic regionlocated in exon 1 are probes comprising a nucleotide sequence set forthin any of SEQ ID NO: 98-105; probes for detecting specific allelicvariants of the polymorphic region located in exon 3 are probescomprising a nucleotide sequence set forth in any of SEQ ID NO: 106-113;and probes for detecting specific allelic variants of the polymorphicregion located in exon 8 are probes comprising a nucleotide sequence setforth in any of SEQ ID NO: 67-74. Examples of probes for detectingspecific allelic variants of the polymorphic region located in intron 5are probes comprising a nucleotide sequence set forth in any of SEQ IDNO: 75-82; and probes for detecting specific allelic variants of thepolymorphic region located in intron 10 are probes comprising anucleotide sequence set forth in any of SEQ ID NO: 114-121. In apreferred embodiment of the invention, several probes capable ofhybridizing specifically to allelic variants are attached to a solidphase support, e.g, a “chip”. Oligonucleotides can be bound to a solidsupport by a variety of processes, including lithography. For example achip can hold up to 250,000 oligonucleotides (GeneChip, Affymetrix).Mutation detection analysis using these chips comprisingoligonucleotides, also termed “DNA probe arrays” is described e.g., inCronin et al. (1996) Human Mutation 7:244. In one embodiment, a chipcomprises all the allelic variants of at least one polymorphic region ofa gene. The solid phase support is then contacted with a test nucleicacid and hybridization to the specific probes is detected. Accordingly,the identity of numerous allelic variants of one or more genes can beidentified in a simple hybridization experiment. For example, theidentity of the allelic variant of the nucleotide polymorphism in exons1, 3, 8 or in introns 5 and 10 can be determined in a singlehybridization experiment.

[0178] In other detection methods, it is necessary to first amplify atleast a portion of an SR-BI gene prior to identifying the allelicvariant. Amplification can be performed, e.g., by PCR and/or LCR,according to methods known in the art. In one embodiment, genomic DNA ofa cell is exposed to two PCR primers and amplification for a number ofcycles sufficient to produce the required amount of amplified DNA. Inpreferred embodiments, the primers are located between 150 and 350 basepairs apart. Preferred primers, such as primers for amplifying each ofthe exons of the human SR-BI gene, are listed in Table IX in theExamples. Details regarding the PCR reaction are indicated in TableVIII, also in the Examples.

[0179] Alternative amplification methods include: self sustainedsequence replication (Guatelli, J. C. et al., 1990, Proc. Natl. Acad.Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D.Y. et al., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-BetaReplicase (Lizardi, P. M. et al., 1988, Bio/Technology 6:1197), or anyother nucleic acid amplification method, followed by the detection ofthe amplified molecules using techniques well known to those of skill inthe art. These detection schemes are especially useful for the detectionof nucleic acid molecules if such molecules are present in very lownumbers.

[0180] In one embodiment, any of a variety of sequencing reactions knownin the art can be used to directly sequence at least a portion of anSR-BI gene and detect allelic variants, e.g, mutations, by comparing thesequence of the sample sequence with the corresponding wild-type(control) sequence. Exemplary sequencing reactions include those basedon techniques developed by Maxam and Gilbert (Proc. Natl Acad Sci USA(1977) 74:560) or Sanger (Sanger et al (1977) Proc. Nat. Acad. Sci74:5463). It is also contemplated that any of a variety of automatedsequencing procedures may be utilized when performing the subject assays(Biotechniques (1995) 19:448), including sequencing by mass spectrometry(see, for example, U.S. Pat. No. 5,547,835 and international patentapplication Publication Number WO 94/16101, entitled DNA Sequencing byMass Spectrometry by H. Köster; U.S. Pat. No. 5,547,835 andinternational patent application Publication Number WO 94/21822 entitled“DNA Sequencing by Mass Spectrometry Via Exonuclease Degradation” by H.Köster), and U.S. Pat. No. 5,605,798 and International PatentApplication No. PCT/US96/03651 entitled DNA Diagnostics Based on MassSpectrometry by H. Köster;. Cohen et al. (1996) Adv Chromatogr36:127-162; and Griffin et al. (1993) Appl Biochem Biotechnol38:147-159). It will be evident to one skilled in the art that, forcertain embodiments, the occurrence of only one, two or three of thenucleic acid bases need be determined in the sequencing reaction. Forinstance, A-track or the like, e.g., where only one nucleotide isdetected, can be carried out.

[0181] Yet other sequencing methods are disclosed, e.g., in U.S. Pat.No. 5,580,732 entitled “Method of DNA sequencing employing a mixedDNA-polymer chain probe” and U.S. Pat. No. 5,571,676 entitled “Methodfor mismatch-directed in vitro DNA sequencing”.

[0182] In some cases, the presence of a specific allele of an SR-BI genein DNA from a subject can be shown by restriction enzyme analysis. Forexample, a specific nucleotide polymorphism can result in a nucleotidesequence comprising a restriction site which is absent from thenucleotide sequence of another allelic variant. In particular, thepresence of a cytidine at position 54 of intron 5 creates an Apal site,whereas the presence of a thymidine, at this position destroys the Apalsite. Similarly, the polylmorphism of exon 1 and exon 8 can bedetermined by analyzing the products or restriction digests (see TableIX).

[0183] In a further embodiment, protection from cleavage agents (such asa nuclease, hydroxylamine or osmium tetroxide and with piperidine) canbe used to detect mismatched bases in RNA/RNA DNA/DNA, or RNA/DNAheteroduplexes (Myers, et al. (1985) Science 230:1242). In general, thetechnique of “mismatch cleavage” starts by providing heteroduplexesformed by hybridizing a control nucleic acid, which is optionallylabeled, e.g., RNA or DNA, comprising a nucleotide sequence of an SR-BIallelic variant with a sample nucleic acid, e.g, RNA or DNA, obtainedfrom a tissue sample. The double-stranded duplexes are treated with anagent which cleaves single-stranded regions of the duplex such asduplexes formed based on basepair mismatches between the control andsample strands. For instance, RNA/DNA duplexes can be treated with RNaseand DNA/DNA hybrids treated with S1 nuclease to enzymatically digest themismatched regions. In other embodiments, either DNA/DNA or RNA/DNAduplexes can be treated with hydroxylamine or osmium tetroxide and withpiperidine in order to digest mismatched regions. After digestion of themismatched regions, the resulting material is then separated by size ondenaturing polyacrylamide gels to determine whether the control andsample nucleic acids have an identical nucleotide sequence or in whichnucleotides they are different. See, for example, Cotton et al (1988)Proc. Natl Acad Sci USA 85:4397; Saleeba et al (1992) Methods Enzymod.217:286-295. In a preferred embodiment, the control or sample nucleicacid is labeled for detection.

[0184] In other embodiments, alterations in electrophoretic mobility isused to identify the type of SR-BI allelic variant. For example, singlestrand conformation polymorphism (SSCP) may be used to detectdifferences in electrophoretic mobility between mutant and wild typenucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA 86:2766, seealso Cotton (1993) Mutat Res 285:125-144; and Hayashi (1992) Genet AnalTech Appl 9:73-79). Single-stranded DNA fragments of sample and controlnucleic acids are denatured and allowed to renature. The secondarystructure of single-stranded nucleic acids varies according to sequence,the resulting alteration in electrophoretic mobility enables thedetection of even a single base change. The DNA fragments may be labeledor detected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In anotherpreferred embodiment, the subject method utilizes heteroduplex analysisto separate double stranded heteroduplex molecules on the basis ofchanges in electrophoretic mobility (Keen et al. (1991) Trends Genet7:5).

[0185] In yet another embodiment, the identity of an allelic variant ofa polymorphic region is obtained by analyzing the movement of a nucleicacid comprising the polymorphic region in polyacrylamide gels containinga gradient of denaturant is assayed using denaturing gradient gelelectrophoresis (DGGE) (Myers et al (1985) Nature 313:495). When DGGE isused as the method of analysis, DNA will be modified to insure that itdoes not completely denature, for example by adding a GC clamp ofapproximately 40 bp of high-melting GC-rich DNA by PCR. In a furtherembodiment, a temperature gradient is used in place of a denaturingagent gradient to identify differences in the mobility of control andsample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:1275).

[0186] Examples of techniques for detecting differences of at least onenucleotide between 2 nucleic acids include, but are not limited to,selective oligonucleotide hybridization, selective amplification, orselective primer extension. For example, oligonucleotide probes may beprepared in which the known polymorphic nucleotide is placed centrally(allele-specific probes) and then hybridized to target DNA underconditions which permit hybridization only if a perfect match is found(Saiki et al. (1986) Nature 324:163); Saiki et al (1989) Proc. NatlAcad. Sci USA 86:6230; and Wallace et al. (1979) Nucl. Acids Res.6:3543). Such allele specific oligonucleotide hybridization techniquesmay be used for the simultaneous detection of several nucleotide changesin different polylmorphic regions of SR-BI. For example,oligonucleotides having nucleotide sequences of specific allelicvariants are attached to a hybridizing membrane and this membrane isthen hybridized with labeled sample nucleic acid. Analysis of thehybridization signal will then reveal the identity of the nucleotides ofthe sample nucleic acid.

[0187] Alternatively, allele specific amplification technology whichdepends on selective PCR amplification may be used in conjunction withthe instant invention. Oligonucleotides used as primers for specificamplification may carry the allelic variant of interest in the center ofthe molecule (so that amplification depends on differentialhybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) orat the extreme 3′ end of one primer where, under appropriate conditions,mismatch can prevent, or reduce polymerase extension (Prossner (1993)Tibtech 11:238; Newton et al. (1989) Nucl. Acids Res. 17:2503). Thistechnique is also termed “PROBE” for Probe Oligo Base Extension. Inaddition it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al (1992) Mol. Cell Probes 6:1).

[0188] In another embodiment, identification of the allelic variant iscarried out using an oligonucleotide ligation assay (OLA), as described,e.g., in U.S. Pat. No. 4,998,617 and in Landegren, U. et al., Science241:1077-1080 (1988). The OLA protocol uses two oligonucleotides whichare designed to be capable of hybridizing to abutting sequences of asingle strand of a target. One of the oligonucleotides is linked to aseparation marker, e.g., biotinylated, and the other is detectablylabeled. If the precise complementary sequence is found in a targetmolecule, the oligonucleotides will hybridize such that their terminiabut, and create a ligation substrate. Ligation then permits the labeledoligonucleotide to be recovered using avidin, or another biotin ligand.Nickerson, D. A. et al. have described a nucleic acid detection assaythat combines attributes of PCR and OLA (Nickerson, D. A. et al., Proc.Natl. Acad. Sci. (U.S.A.) 87:8923-8927 (1990). In this method, PCR isused to achieve the exponential amplification of target DNA, which isthen detected using OLA.

[0189] Several techniques based on this OLA method have been developedand can be used to detect specific allelic variants of a polymorphicregion of an SR-BI gene. For example, U.S. Pat. No. 5593826 discloses anOLA using an oligonucleotide having 3′-amino group and a5′-phosphorylated oligonucleotide to form a conjugate having aphosphoramidate linkage. In another variation of OLA described in Tobeet al. ((1996)Nucleic Acids Res 24: 3728), OLA combined with PCR permitstyping of two alleles in a single microtiter well. By marking each ofthe allele-specific primers with a unique hapten, i.e. digoxigenin andfluorescein, each OLA reaction can be detected by using hapten specificantibodies that are labeled with different enzyme reporters, alkalinephosphatase or horseradish peroxidase. This system permits the detectionof the two alleles using a high throughput format that leads to theproduction of two different colors.

[0190] The invention further provides methods for detecting singlenucleotide polymorphisms in an SR-BI gene. Because single nucleotidepolymorphisms constitute sites of variation flanked by regions ofinvariant sequence, their analysis requires no more than thedetermination of the identity of the single nucleotide present at thesite of variation and it is unnecessary to determine a complete genesequence for each patient. Several methods have been developed tofacilitate the analysis of such single nucleotide polymorphisms.

[0191] In one embodiment, the single base polymorphism can be detectedby using a specialized exonuclease-resistant nucleotide, as disclosed,erg., in Mundy, C. R. (U.S. Pat. No. 4,656,127). According to themethod, a primer complementary to the allelic sequence immediately 3′ tothe polymorphic site is permitted to hybridize to a target moleculeobtained from a particular animal or human. If the polymorphic site onthe target molecule contains a nucleotide that is complementary to theparticular exonuclease-resistant nucleotide derivative present, thenthat derivative will be incorporated onto the end of the hybridizedprimer. Such incorporation renders the primer resistant to exonuclease,and thereby permits its detection. Since the identity of theexonuclease-resistant derivative of the sample is known, a finding thatthe primer has become resistant to exonucleases reveals that thenucleotide present in the polymorphic site of the target molecule wascomplementary to that of the nucleotide derivative used in the reaction.This method has the advantage that it does not require the determinationof large amounts of extraneous sequence data.

[0192] In another embodiment of the invention, a solution-based methodis used for determining the identity of the nucleotide of a polymorphicsite. Cohen, D. et al. (French Patent 2,650,840; PCT Appln. No.WO91/02087). As in the Mundy method of U.S. Pat. No. 4,656,127, a primeris employed that is complementary to allelic sequences immediately 3′ toa polymorphic site. The method determines the identity of the nucleotideof that site using labeled dideoxynucleotide derivatives, which, ifcomplementary to the nucleotide of the polymorphic site will becomeincorporated onto the terminus of the primer.

[0193] An alternative method, known as Genetic Bit Analysis or GBA isdescribed by Goelet, P. et al. (PCT Appln. No. 92/15712). The method ofGoelet, P. et al. uses mixtures of labeled terminators and a primer thatis complementary to the sequence 3′ to a polymorphic site. The labeledterminator that is incorporated is thus determined by, and complementaryto, the nucleotide present in the polymorphic site of the targetmolecule being evaluated. In contrast to the method of Cohen et al.(French Patent 2,650,840; PCT Appln. No. WO91/02087) the method ofGoelet, P. et al. is preferably a heterogeneous phase assay, in whichthe primer or the target molecule is immobilized to a solid phase.

[0194] Recently, several primer-guided nucleotide incorporationprocedures for assaying polymorphic sites in DNA have been described(Komher, J. S. et al., Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov,B. P., Nucl. Acids Res. 18:3671 (1990); Syvanen, A. −C., et al.,Genomics 8:684-692 (1990); Kuppuswamy, M. N. et al., Proc. Natl. Acad.Sci. (U.S.A.) 88:1143-1147 (1991); Prezant, T. R. et al., Hum. Mutat.1:159-164 (1992); Ugozzoli, L. et al., GATA 9:107-112 (1992); Nyren, P.et al., Anal. Biochem. 208:171-175 (1993)). These methods differ fromGBA in that they all rely on the incorporation of labeleddeoxynucleotides to discriminate between bases at a polymorphic site. Insuch a format, since the signal is proportional to the number ofdeoxynucleotides incorporated, polymorphisms that occur in runs of thesame nucleotide can result in signals that are proportional to thelength of the run (Syvanen, A. −C., et al., Amer. J. Hum. Genet.52:46-59 (1993)).

[0195] For determining the identity of the allelic variant of apolymorphic region located in the coding region of an SR-BI gene, yetother methods than those described above can be used. For example,identification of an allelic variant which encodes a mutated SR-BIprotein can be performed by using an antibody specifically recognizingthe mutant protein in, e.g., immunohistochemistry orimmunoprecipitation. Antibodies to wild-type SR-BI protein aredescribed, e.g, in Acton et al. (1999) Science 271:518 (anti-mouse SR-BIantibody cross-reactive with human SR-BI). Other antibodies to wild-typeSR-BI or mutated forms of SR-BI proteins can be prepared according tomethods known in the art. Preferred antibodies specifically bind to ahuman SR-BI protein having a serine at residue 2 and/or having anisoleucine at amino acid residue 135. Alternatively, one can alsomeasure an activity of an SR-BI protein, such as binding to a lipid orlipoprotein. Binding assays are known in the art, and involve, e.g.,obtaining cells from a subject, and performing binding experiments witha labeled lipid, to determine whether binding to the mutated form of thereceptor differs from binding to the wild-type of the receptor.

[0196] Antibodies directed against wild type or mutant SR-BIpolypeptides or allelic variant thereof, which are discussed above, mayalso be used in disease diagnostics and prognostics. Such diagnosticmethods, may be used to detect abnormalities in the level of SR-BIpolypeptide expression, or abnormalities in the structure and/or tissue,cellular, or subcellular location of an SR-BI polypeptide. Structuraldifferences may include, for example, differences in the size,electronegativity, or antigenicity of the mutant SR-BI polypeptiderelative to the normal SR-BI polypeptide. Protein from the tissue orcell type to be analyzed may easily be detected or isolated usingtechniques which are well known to one of skill in the art, includingbut not limited to western blot analysis. For a detailed explanation ofmethods for carrying out Western blot analysis, see Sambrook et al,1989, supra, at Chapter 18. The protein detection and isolation methodsemployed herein may also be such as those described in Harlow and Lane,for example, (Harlow, E. and Lane, D., 1988, “Antibodies: A LaboratoryManual”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.),which is incorporated herein by reference in its entirety.

[0197] This can be accomplished, for example, by immunofluorescencetechniques employing a fluorescently labeled antibody (see below)coupled with light microscopic, flow cytometric, or fluorimetricdetection. The antibodies (or fragments thereof) useful in the presentinvention may, additionally, be employed histologically, as inimmunofluorescence or immunoelectron microscopy, for in situ detectionof SR-BI polypeptides. In situ detection may be accomplished by removinga histological specimen from a patient, and applying thereto a labeledantibody of the present invention. The antibody (or fragment) ispreferably applied by overlaying the labeled antibody (or fragment) ontoa biological sample. Through the use of such a procedure, it is possibleto determine not only the presence of the SR-BI polypeptide, but alsoits distribution in the examined tissue. Using the present invention,one of ordinary skill will readily perceive that any of a wide varietyof histological methods (such as staining procedures) can be modified inorder to achieve such in situ detection.

[0198] Often a solid phase support or carrier is used as a supportcapable of binding an antigen or an antibody. Well-known supports orcarriers include glass, polystyrene, polypropylene, polyethylene,dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, gabbros, and magnetite. The nature of the carrier canbe either soluble to some extent or insoluble for the purposes of thepresent invention. The support material may have virtually any possiblestructural configuration so long as the coupled molecule is capable ofbinding to an antigen or antibody. Thus, the support configuration maybe spherical, as in a bead, or cylindrical, as in the inside surface ofa test tube, or the external surface of a rod. Alternatively, thesurface may be flat such as a sheet, test strip, etc. Preferred supportsinclude polystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

[0199] One means for labeling an anti-SR-BI polypeptide specificantibody is via linkage to an enzyme and use in an enzyme immunoassay(EIA) (Voller, “The Enzyme Linked Immunosorbent Assay (ELISA)”,Diagnostic Horizons 2:1-7, 1978, Microbiological Associates QuarterlyPublication, Walkersville, MD; Voller, et al., J. Clin. Pathol.31:507-520 (1978); Butler, Meth. Enzymol. 73:482-523 (1981); Maggio,(ed.) Enzyme Immunoassay, CRC Press, Boca Raton, Fla., 1980; Ishikawa,et al., (eds.) Enzyme Immunoassay, Kgaku Shoin, Tokyo, 1981). The enzymewhich is bound to the antibody will react with an appropriate substrate,preferably a chromogenic substrate, in such a manner as to produce achemical moiety which can be detected, for example, byspectrophotometric, fluorimetric or by visual means. Enzymes which canbe used to detectably label the antibody include, but are not limitedto, malate dehydrogenase, staphylococcal nuclease, delta-5-steroidisomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate,dehydrogenase, triose phosphate isomerase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoamylase and acetylcholinesterase. The detection can be accomplishedby colorimetric methods which employ a chromogenic substrate for theenzyme. Detection may also be accomplished by visual comparison of theextent of enzymatic reaction of a substrate in comparison with similarlyprepared standards.

[0200] Detection may also be accomplished using any of a variety ofother immunoassays. For example, by radioactively labeling theantibodies or antibody fragments, it is possible to detect fingerprintgene wild type or mutant peptides through the use of a radioimmunoassay(RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays,Seventh Training Course on Radioligand Assay Techniques, The EndocrineSociety, March 1986, which is incorporated by reference herein). Theradioactive isotope can be detected by such means as the use of a gammacounter or a scintillation counter or by autoradiography.

[0201] It is also possible to label the antibody with a fluorescentcompound. When the fluorescently labeled antibody is exposed to light ofthe proper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

[0202] The antibody can also be detectably labeled using fluorescenceemitting metals such as ¹⁵²Eu, or others of the lanthanide series. Thesemetals can be attached to the antibody using such metal chelating groupsas diethylenetriaminepentacetic acid (DTPA) orethylenediaminetetraacetic acid (EDTA).

[0203] The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

[0204] Likewise, a bioluminescent compound may be used to label theantibody of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems in, which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Important bioluminescent compounds forpurposes of labeling are luciferin, luciferase and aequorin.

[0205] Moreover, it will be understood that any of the above methods fordetecting alterations in a gene or gene product or polymorphic variantscan be used to monitor the course of treatment or therapy.

[0206] If a polymorphic region is located in an exon, either in a codingor non-coding portion of the gene, the identity of the allelic variantcan be determined by determining the molecular structure of the mRNA,pre-mRNA, or cDNA. The molecular structure can be determined using anyof the above described methods for determining the molecular structureof the genomic DNA, e.g., sequencing and SSCP.

[0207] The methods described herein may be performed, for example, byutilizing pre-packaged diagnostic kits, such as those described above,comprising at least one probe or primer nucleic acid described herein,which may be conveniently used, e.g., to determine whether a subject hasor is at risk of developing a disease associated with a specific SR-BIallelic variant.

[0208] Sample nucleic acid for using in the above-described diagnosticand prognostic methods can be obtained from any cell type or tissue of asubject. For example, a subject's bodily fluid (e.g, blood) can beobtained by known techniques (e.g., venipuncture). Alternatively,nucleic acid tests can be performed on dry samples (e.g., hair or skin).Fetal nucleic acid samples can be obtained from maternal blood asdescribed in International Patent Application No. WO91/07660 to Bianchi.Alternatively, amniocytes or chorionic villi may be obtained forperforming prenatal testing.

[0209] Diagnostic procedures may also be performed in situ directly upontissue sections (fixed and/or frozen) of patient tissue obtained frombiopsies or resections, such that no nucleic acid purification isnecessary. Nucleic acid reagents may be used as probes and/or primersfor such in situ procedures (see, for example, Nuovo, G. J., 1992, PCRin situ hybridization: protocols and applications, Raven Press, N.Y.).

[0210] In addition to methods which focus primarily on the detection ofone nucleic acid sequence, profiles may also be assessed in suchdetection schemes. Fingerprint profiles may be generated, for example,by utilizing a differential display procedure, Northern analysis and/orRT-PCR.

[0211] Pharmacogenomics

[0212] Knowledge of the identity of the allele of one or more SR-BI genepolymorphic regions in an individual (the SR-BI genetic profile), aloneor in conjunction with information on other genetic defects contributingto the same disease (the genetic profile of the particular disease)allows a customization of the therapy for a particular disease to theindividual's genetic profile, the goal of “pharmacogenomics”. Forexample, subjects having a specific allele of an SR-BI gene may or maynot exhibit symptoms of a particular disease or be predisposed todeveloping symptoms of a particular disease. Further, if those subjectsare symptomatic, they may or may not respond to a certain drug, e.g., aspecific SR-BI therapeutic, but may respond to another. Thus, generationof an SR-BI genetic profile, (e.g., categorization of alterations inSR-BI genes which are associated with the development of a particulardisease), from a population of subjects, who are symptomatic for adisease or condition that is caused by or contributed to by a defectiveand/or deficient SR-BI gene and/or protein (an SR-BI genetic populationprofile) and comparison of an individual's SR-BI profile to thepopulation profile, permits the selection or design of drugs that areexpected to be safe and efficacious for a particular patient or patientpopulation (i.e., a group of patients having the same geneticalteration).

[0213] For example, an SR-BI population profile can be performed bydetermining the SR-BI profile, e.g., the identity of SR-BI alleles, in apatient population having a disease, which is associated with one ormore specific alleles of SR-BI polymorphic regions. Optionally, theSR-BI population profile can further include information relating to theresponse of the population to an SR-BI therapeutic, using any of avariety of methods, including, monitoring: 1) the severity of symptomsassociated with the SR-BI related disease, 2) SR-BI gene expressionlevel, 3) SR-BI mRNA level, and/or 4) SR-BI protein level. and (iii)dividing or categorizing the population based on particular SR-BIalleles. The SR-BI genetic population profile can also, optionally,indicate those particular SR-BI alleles which are present in patientsthat are either responsive or non-responsive to a particulartherapeutic. This information or population profile, is then useful forpredicting which individuals should respond to particular drugs, basedon their individual SR-BI profile.

[0214] In a preferred embodiment, the SR-BI profile is a transcriptionalor expression level profile and step (i) is comprised of determining theexpression level of SR-BI proteins, alone or in conjunction with theexpression level of other genes known to contribute to the same diseaseat various stages of the disease.

[0215] Pharmacogenomic studies can also be performed using transgenicanimals. For example, one can produce transgenic mice, e.g., asdescribed herein, which contain a specific allelic variant of an SR-BIgene. These mice can be created, e.g., by replacing their wild-typeSR-BI gene with an allele of the human SR-BI gene. The response of thesemice to specific SR-BI therapeutics can then be determined.

[0216] Monitoring Effects of SR-BI Therapeutics During Clinical Trials

[0217] The ability to target populations expected to show the highestclinical benefit, based on the SR-BI or disease genetic profile, canenable: 1) the repositioning of marketed drugs with disappointing marketresults; 2) the rescue of drug candidates whose clinical development hasbeen discontinued as a result of safety or efficacy limitations, whichare patient subgroup-specific; and 3) an accelerated and less costlydevelopment for drug candidates and more optimal drug labeling (e.g.since the use of SR-BI as a marker is useful for optimizing effectivedose).

[0218] In situations in which the disease associated with a specificSR-BI allele is characterized by an abnormal SR-BI expression, thetreatment of an individual with an SR-BI therapeutic can be monitored bydetermining SR-BI characteristics, such as SR-BI protein level oractivity, SR-BI mRNA level, and/or SR-BI transcriptional level. Thismeasurement will indicate whether the treatment is effective or whetherit should be adjusted or optimized. Thus, SR-BI can be used as a markerfor the efficacy of a drug during clinical trials.

[0219] In a preferred embodiment, the present invention provides amethod for monitoring the effectiveness of treatment of a subject withan agent (e.g., an agonist, antagonist, peptidomimetic, protein,peptide, nucleic acid, small molecule, or other drug candidateidentified by the screening assays described herein) comprising thesteps of (i) obtaining a preadministration sample from a subject priorto administration of the agent; (ii) detecting the level of expressionof an SR-BI protein, mRNA, or genomic DNA in the preadministrationsample; (iii) obtaining one or more post-administration samples from thesubject; (iv) detecting the level of expression or activity of the SR-BIprotein, mRNA, or genomic DNA in the post-administration samples; (v)comparing the level of expression or activity of the SR-BI protein,mRNA, or genomic DNA in the preadministration sample with the SR-BIprotein, mRNA, or genomic DNA in the post administration sample orsamples; and (vi) altering the administration of the agent to thesubject accordingly. For example, increased administration of the agentmay be desirable to increase the expression or activity of SR-BI tohigher levels than detected, i.e., to increase the effectiveness of theagent. Alternatively, decreased administration of the agent may bedesirable to decrease expression or activity of SR-BI to lower levelsthan detected, i.e., to decrease the effectiveness of the agent.

[0220] Cells of a subject may also be obtained before and afteradministration of an SR-BI therapeutic to detect the level of expressionof genes other than SR-BI, to verify that the SR-BI therapeutic does notincrease or decrease the expression of genes which could be deleterious.This can be done, e.g., by using the method of transcriptionalprofiling. Thus, mRNA from cells exposed in vivo to an SR-BI therapeuticand mRNA from the same type of cells that were not exposed to the SR-BItherapeutic could be reverse transcribed and hybridized to a chipcontaining DNA from numerous genes, to thereby compare the expression ofgenes in cells treated and not treated with an SR-BI therapeutic. If,for example an SR-BI therapeutic turns on the expression of aproto-oncogene in an individual, use of this particular SR-BItherapeutic may be undesirable.

[0221] Methods of Treatment

[0222] The present invention provides for both prophylactic andtherapeutic methods of treating a subject having or likely to develop adisorder associated with specific SR-BI alleles and/or aberrant SR-BIexpression or activity, e.g., disorders or diseases associated with anabnormal BMI or lipid levels such as cardiovascular disease or diabetes.

[0223] Prophylactic Methods

[0224] In one aspect, the invention provides a method for preventing ina subject, a disease or condition associated with a specific SR-BIallele and/or an aberrant SR-BI expression or activity, such as a bodymass disorder or abnormal lipid level and medical conditions resultingtherefrom, by administering to the subject an agent which counteractsthe unfavorable biological effect of the specific SR-BI allele. Subjectsat risk for such a disease can be identified by a diagnostic orprognostic assay, e.g., as described herein. Administration of aprophylactic agent can occur prior to the manifestation of symptomsassociated with specific SR-BI alleles, such that a disease or disorderis prevented or, alternatively, delayed in its progression. Depending onthe identity of the SR-BI allele in a subject, a compound thatcounteracts the effect of this allele is administered. The compound canbe a compound modulating the plasma level of lipids. The treatment canalso be a specific diet. In particular, the treatment can be undertakenprophylactically, before any other symptoms are present. Such aprophylactic treatment could thus prevent the development of an abnormalBMI or lipid level, e.g., abnormally high LDL level or abnormally lowHDL. The prophylactic methods are similar to therapeutic methods of thepresent invention and are further discussed in the followingsubsections.

[0225] Therapeutic Methods

[0226] The invention further provides methods of treating subjectshaving a disease or disorder associated with a specific allelic variantof a polymorphic region of an SR-BI gene. Preferred diseases ordisorders include those associated with an abnormal body mass, orabnormal lipoprotein (LDL and HDL) levels and disorders resultingtherefrom (e.g. cardiovascular disease, obesity, cachexia, diabetes, andgallstone formation). In one embodiment, the method comprises (a)determining the identity of the allelic variant; and (b) administeringto the subject a compound that compensates for the effect of thespecific allelic variant. The polymorphic region can be localized at anylocation of the gene, e.g., in the promoter (e.g., in a regulatoryelement of the promoter), in an exon, (e.g., coding region of an exon),in an intron, or at an exon/intron border. Thus, depending on the siteof the polymorphism in the SR-BI gene, a subject having a specificvariant of the polymorphic region which is associated with a specificdisease or condition, can be treated with compounds which specificallycompensate for the allelic variant.

[0227] In a preferred embodiment, the identity of one or more of thefollowing nucleotides of an SR-BI gene of a subject is determined:nucleotide 146 of exon 1, nucleotide 119 of exon 3, nucleotide 41 ofexon 8, nucleotide 54 of intron 5, and nucleotide −41 of intron 10. If afemale subject has the more common allele of residue 41 of exon 8(EX8C), high LDL levels and resulting cardiovascular disorders can betreated, prevented from occurring or can be reduced by administering tothe subject a pharmaceutically effective amount of a compound to reduceLDL level to a normal LDL level. Similarly, if a female subject has theless common allele of residue 54 of intron 5 (IVS5T), a high BMI and/orLDL level and consequences thereof, such as diabetes and cardiovasculardisorders, can be treated, prevented from occurring or can be reduced,by administering to the subject a pharmaceutically effective amount of acompound to reduce the BMI and/or the LDL levels. In another embodimentof the invention, if a male subject has the more common allele atresidue 41 of exon 8 (EX8C), the more common allele at residue 54 ofintron 5 (IVS5C), and the more common allele at residue 146 of exon 1,development of low HDL levels can be treated, prevented or increased byadministering to the subject a pharmaceutically effective amount of acompound that increases HDL levels, thereby preventing resultingcardiovascular disorders. Likewise, if a female or a male subject hasthe more common allele at residue 41 of exon 8 (EX8C), development oflow HDL levels and resulting cardiovascular disorders can be treated orprevented from occurring by administering to the subject apharmaceutically effective amount of a compound which increases HDLlevel to a normal HDL level. Similarly, if a female subject has the lesscommon allele of residue 54 of intron 5 (IVS5T), low HDL and resultingcardiovascular disorders can be treated or prevented from occurring, byadministering to the subject a pharmaceutically effective amount of acompound which increases HDL level to a normal HDL level. Furthermore,if a female subject has both the more common allele at residue 41 ofexon 8 (EX8C) and the less common allele of residue 54 of intron 5(IVS5T), low HDL levels and resulting cardiovascular disorders can betreated or prevented from occurring, by administering to the subject apharmaceutically effective amount of a compound which increases HDLlevel to a normal HDL level. In addition, in a female subject, theidentification of specific alleles within the SR-B1 gene (e.g., EX8 orIVS5) can be used to predict the effect of HRT on lipid levels (e.g.,HDL levels).

[0228] Generally, the allelic variant can be a mutant allele, i.e., anallele which when present in one, or preferably two copies, in a subjectresults in a change in the phenotype of the subject. A mutation can be asubstitution, deletion, and/or addition of at least one nucleotiderelative to the wild-type allele. Depending on where the mutation islocated in the SR-BI gene, the subject can be treated to specificallycompensate for the mutation. For example, if the mutation is present inthe coding region of the gene and results in an inactive or less activeSR-BI protein, the subject can be treated, e.g., by administration tothe subject of a nucleic acid encoding a wild-type SR-BI protein, suchthat the expression of the wild-type SR-BI protein compensates for theendogenous mutated form of the SR-BI protein. Nucleic acids encodingwild-type human SR-BI protein are set forth in SEQ ID Nos. 1 and 3 andare described, e.g., in Calvo and Vega (1993) J. Biol. Chem. 268:18929.

[0229] Furthermore, depending on the site of the mutation in the SR-BIprotein and the specific effect on its activity, specific treatments canbe designed to compensate for that effect. The SR-BI protein is a cellsurface receptor which binds specific forms of lipids, e.g., modifiedlipid or lipoproteins, e.g., HDL. Thus, an SR-BI protein has anextracellular domain which binds specific molecules, e.g., lipids, atransmembrane domain, and an intracellular domain, which is likely totransmit an intracellular signal. The structure of SR-BI proteins isfurther described, e.g., in Calvo and Vega, supra; Acton et al. (1994)J. Biol. Chem. 269:21003; Acton et al. (1995) Science 271:518; Rigottiet al. (1995) J. Biol. Chem. 270:16221; Fukasawa et al. (1996) Exp.Cell. Res. 222:246; Wang et al. (1996) J. Biol. Chem. 271:21001; andpublished PCT Application having publication number WO 96/00288 by Actonet al. Thus, if the mutation results in an SR-BI protein which is lesscapable of binding certain types of modified lipids, resulting in anaccumulation of such lipids in the subject, a treatment can be designedwhich removes such modified lipids from the subject. In one embodiment,a compound which binds this form of lipid and is capable of targetingthe lipid to a site where it is eliminated, is administered to thesubject. Alternatively, the expression of another cell surface receptorwhich binds this type of lipid can be increased. In fact, both SR-BI andthe class B scavenger receptor CD36 are capable of interacting withanionic phospholipids (Rigotti et al., supra). Thus, if a subject has amutant SR-BI protein which is defective in its binding to anionicphospholipids, the subject can be treated by administration of acompound which increases CD36 protein levels in the cells.

[0230] In situations in which the mutant SR-BI protein binds certainforms of lipids with higher affinity, and if this is causing orcontributing to a disease, a subject having such a mutated SR-BI proteincan be treated, e.g., by administration of compounds which inhibit ordecrease the interaction between the specific form of the lipid andSR-BI. For example, soluble forms of SR-BI proteins or binding fragmentsthereof, can be administered to the subject. Alternatively, smallmolecules can be administered to the subject for interfering in theinteraction between SR-BI and a lipid.

[0231] A mutant SR-BI protein can also be an SR-BI protein having amutation in the cytoplasmic domain of the protein which results in anaberrant signal transduction from the receptor. Subjects having such amutation can be treated, e.g., by administration of compounds whichinduce the same or similar signal transduction or compounds which actdownstream of the receptor.

[0232] The effect of a mutation in an SR-BI protein can be determinedaccording to methods known in the art. For example, if the mutation islocated in the extracellular portion of the protein, one can performbinding assays with specific forms of lipids, e.g., HDL, and determinewhether the binding affinity of such lipid with the mutated SR-BIprotein is different from the binding affinity of the lipid with thewild-type protein. Such assays can be performed using a soluble form ofan SR-BI protein or a membrane bound form of the protein. If themutation in the SR-BI protein is located in the cytoplasmic domain ofthe protein, signal transduction experiments can be performed todetermine whether the signal transduced from the mutated receptordiffers from the signal transduced from the wild-type receptor.Alternatively, one can also investigate whether binding to a proteinwhich interacts with the cytoplasmic domain of the receptor is affectedby the mutation. Such determination can be made by, e.g., byimmunoprecipitation.

[0233] Yet in another embodiment, the invention provides methods fortreating a subject having a mutated SR-BI gene, in which the mutation islocated in a regulatory region of the gene. Such a regulatory region canbe localized in the promoter of the gene, in the 5′ or 3′ untranslatedregion of an exon, or in an intron. A mutation in a regulatory regioncan result in increased production of SR-BI protein, decreasedproduction of SR-BI protein, or production of SR-BI having an aberranttissue distribution. The effect of a mutation in a regulatory regionupon the SR-BI protein can be determined, e.g., by measuring the SR-BIprotein level or mRNA level in cells having an SR-BI gene having thismutation and which, normally (i.e., in the absence of the mutation)produce SR-BI protein. The effect of a mutation can also be determinedin vitro. For example, if the mutation is in the promoter, a reporterconstruct can be constructed which comprises the mutated promoter linkedto a reporter gene, the construct transfected into cells, and comparisonof the level of expression of the reporter gene under the control of themutated promoter and under the control of a wild-type promoter. Suchexperiments can also be carried out in mice transgenic for the mutatedpromoter. If the mutation is located in an intron, the effect of themutation can be determined, e.g., by producing transgenic animals inwhich the mutated SR-BI gene has been introduced and in which thewild-type gene may have been knocked out. Comparison of the level ofexpression of SR-BI in the mice transgenic for the mutant human SR-BIgene with mice transgenic for a wild-type human SR-BI gene will revealwhether the mutation results in increased, decreased synthesis of theSR-BI protein and/or aberrant tissue distribution of SR-BI protein. Suchanalysis could also be performed in cultured cells, in which the humanmutant SR-BI gene is introduced and, e.g., replaces the endogenouswild-type SR-BI gene in the cell. Thus, depending on the effect of themutation in a regulatory region of an SR-BI gene, a specific treatmentcan be administered to a subject having such a mutation. Accordingly, ifthe mutation results in decreased production of an SR-BI protein, thesubject can be treated by administration of a compound which increasessynthesis, such as by increasing SR-BI gene expression, and wherein thecompound acts at a regulatory element different from the one which ismutated. Alternatively, if the mutation results in increased SR-BIprotein levels, the subject can be treated by administration of acompound which reduces SR-BI protein production, e.g., by reducing SR-BIgene expression or a compound which inhibits or reduces the activity ofSR-BI.

[0234] Furthermore, it is likely that subjects having different allelicvariants of an SR-BI polymorphic region will respond differently totherapeutic drugs to treat diseases or conditions, such as thoseassociated with an abnormal lipid level. Cholesterol-lowering drugsinclude lovastatin (MEVACOR; Merck & Co.), simvastatin (ZOCOR; Merck &Co.), dextrothyroxine (CHOLOXIN; Knoll Pharmaceutical Co.), pamaqueside(Pfizer), cholestryramine (QUESTRAN; Bristol-Myers Squibb), colestipol(COLESTID; Pharmacia & Upjohn), acipomox (Pharmacia & Upjohn),fenofibrate (LIPIDIL), gemfibrozil (LOPID; Warner-Lambert), cerivastatin(LIPOBAY; Bayer), fluvastatin (LESCOL; Novartis), atorvastatin (LIPITOR,Warner-Lambert), etofylline clofibrate (DUOLIP; Merckle (Germany)),probucol (LORELCO; Hoechst Marion Roussel), omacor (Pronova (Norway),etofibrate (Merz (Germany), clofibrate (ATROMID-S; Wyeth-Ayerst (AHP)),and niacin (numerous manufacturers). Drugs for treating obesity and/orgallstones include dexfenfluramine (REDUX, Interneuron Pharmaceuticals),megestrol acetate (MEGACE, Bristol-Myers Squibb), Phenylpropanolamine(ACUTRIM; Ciba; and DEXUTRIM; Thompson), fluoxetine (PROZAC., Lilly),dextroamphetamine (DEXEDRINE, SmithKline Beecham), fenfluramine andphentermine, chenodiol (CHENIX, Solvay), orlistat (XENICAL, Roche),anandamide (Yissum (Israel)), PCM-4 (Omega Pharmaceutical),mono-octanoin (MOCTAN, Stokely-van Camp), sibutramine (MERIDIA, Knoll),testosterone (TESTODERM, Alza), oxandrolone (OXANDRIN, Bio-TechnologyGeneral), ceruletide diethylamine (TYMTRAN, Pharmacia & Upjohn),testosterone and dihydrotestosterone (ANDROGEL and ANDROGEL-DHT,unimed), somatropin (SEROSTIM, Ares-Serono and BIO-TROPIN, BiotechnologyGeneral), and thalidomide (SYNOVIR, Celgene).

[0235] A correlation between drug responses and specific alleles ofSR-BI can be shown, for example, by clinical studies wherein theresponse to specific drugs of subjects having different allelic variantsof a polymorphic region of an SR-BI gene is compared. Such studies canalso be performed using animal models, such as mice having variousalleles of human SR-BI genes and in which, e.g., the endogenous SR-BIhas been inactivated such as by a knock-out mutation. Test drugs arethen administered to the mice having different human SR-BI alleles andthe response of the different mice to a specific compound is compared.Accordingly, the invention provides assays for identifying the drugwhich will be best suited for treating a specific disease or conditionin a subject. For example, it will be possible to select drugs whichwill be devoid of toxicity, or have the lowest level of toxicitypossible for treating a subject having a disease or condition.

[0236] Computer Readable Means and Arrays

[0237] Computer readable media comprising the allelic variants of thepresent invention is also provided. As used herein, “computer readablemedia” refers to any medium that can be read and accessed directly by acomputer. Such media include, but are not limited to: magnetic storagemedia, such as floppy discs, hard disc storage medium, and magnetictape; optical storage media such as CD-ROM; electrical storage mediasuch as RAM and ROM; and hybrids of these categories such asmagnetic/optical storage media. The skilled artisan will readilyappreciate how any of the presently known computer readable mediums canbe used to create a manufacture comprising computer readable mediumhaving recorded thereon an allelic variant of the present invention.

[0238] As used herein, “recorded” refers to a process for storinginformation on computer readable medium. Those skilled in the art canreadily adopt any of the presently known methods for recordinginformation on computer readable medium to generate manufacturescomprising the allelic variants of the present invention.

[0239] A variety of data processor programs and formats can be used tostore the allelic variant information of the present invention oncomputer readable medium. For example, the nucleic acid sequencecomprising the allelic variant can be represented in a word processingtext file, formatted in commercially-available software such asWordPerfect and MicroSoft Word, or represented in the form of an ASCIIfile, stored in a database application, such as DB2, Sybase, Oracle, orthe like. Any number of dataprocessor structuring formats (e.g., textfile or database) may be adapted in order to obtain computer readablemedium having recorded thereon the allelic variants of the presentinvention.

[0240] By providing the allelic variants of the invention in computerreadable form, one can routinely access the information for a variety ofpurposes. For example, one skilled in the art can use the nucleotide oramino acid sequences of the invention in computer readable form tocompare a target sequence or target structural motif with the sequenceinformation stored within the data storage means. Search means are usedto identify fragments or regions of the sequences of the invention whichmatch a particular target sequence or target motif.

[0241] The invention also includes an array comprising allelic variantsof the present invention. The array can be used to assay expression ofone or more genes in the array. In one embodiment, the array can be usedto assay gene expression in a tissue to ascertain tissue specificity ofgenes in the array. In this manner, up to about 36,000 genes can besimultaneously assayed for expression. This allows a profile to bedeveloped showing a battery of genes specifically expressed in one ormore tissues.

[0242] In addition to such qualitative determination, the inventionallows the quantitation of gene expression. Thus, not only tissuespecificity, but also the level of expression of a battery of genes inthe tissue is ascertainable. Thus, genes can be grouped on the basis oftheir tissue expression per se and level of expression in that tissue.This is useful, for example, in ascertaining the relationship of geneexpression between or among tissues. Thus, one tissue can be perturbedand the effect on gene expression in a second tissue can be determined.In this context, the effect of one cell type on another cell type inresponse to a biological stimulus can be determined. Such adetermination is useful, for example, to know the effect of cell-cellinteraction at the level of gene expression. If an agent is administeredtherapeutically to treat one cell type but has an undesirable effect onanother cell type, the invention provides an assay to determine themolecular basis of the undesirable effect and thus provides theopportunity to co-administer a counteracting agent or otherwise treatthe undesired effect. Similarly, even within a single cell type,undesirable biological effects can be determined at the molecular level.Thus, the effects of an agent on expression of other than the targetgene can be ascertained and counteracted.

[0243] In another embodiment, the array can be used to monitor the timecourse of expression of one or more genes in the array. This can occurin various biological contexts, as disclosed herein, for examplecardiovascular disorders.

[0244] The array is also useful for ascertaining the effect of theexpression of a gene on the expression of other genes in the same cellor in different cells. This provides, for example, for a selection ofalternate molecular targets for therapeutic intervention if the ultimateor downstream target cannot be regulated.

[0245] The array is also useful for ascertaining differential expressionpatterns of one or more genes in normal and abnormal cells. Thisprovides a battery of genes that could serve as a molecular target fordiagnosis or therapeutic intervention.

[0246] Other uses for the nucleic acids of the invention

[0247] The identification of different alleles of SR-BI can also beuseful for identifying an individual among other individuals from thesame species. For example, DNA sequences can be used as a fingerprintfor detection of different individuals within the same species(Thompson, J. S. and Thompson, eds., Genetics in Medicine, WB SaundersCo., Philadelphia, Pa. (1991)). This is useful, e.g., in forensicstudies.

[0248] The present invention is further illustrated by the followingexamples which should not be construed as limiting in any way. Thecontents of all cited references (including literature references,issued patents, published patent applications as cited throughout thisapplication are hereby expressly incorporated by reference. The practiceof the present invention will employ, unless otherwise indicated,conventional techniques of cell biology, cell culture, molecularbiology, transgenic biology, microbiology, recombinant DNA, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature. See, for example, Molecular Cloning ALaboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (ColdSpring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D.N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984);Mullis et al. U.S. Pat. No: 4,683,195; Nucleic Acid Hybridization (B. D.Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D.Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I.Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRLPress, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984);the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); GeneTransfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds.,1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154and 155 (Wu et al. eds.), Immunochemical Methods In Cell And MolecularBiology (Mayer and Walker, eds., Academic Press, London, 1987); HandbookOf Experimental Immunology, Volumes I-IV (D. M. Weir and C. C.Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

EXAMPLES

[0249] The invention now being generally described, it will be morereadily understood by reference to the following examples which areincluded merely for purposes of illustration of certain aspects andembodiments of the present invention, and are not intended to limit theinvention.

Example 1

[0250] Isolation and sequence analysis of genomic DNA encoding the humanSR-BI protein

[0251] A probe consisting of a 474 base pair fragment of the human SR-BIcDNA was used to isolate bacterial artificial chromosomes (BACs)containing genomic DNA encoding the human SR-BI protein from a human BAClibrary (Research Genetics Inc. (Huntsville, Ala.) Cat. #96041)). TwoBACs were isolated by hybridizing the probe to this library. These BACswere then sized by pulse-field electrophoresis and the inserts werefound to be approximately 80 and 70 kilobases long for BAC 179m10 andBAC 256i19, respectively. All further discussion will focus on BAC179m10.

[0252] BAC 179m10 was digested with restriction enzymes and analyzed bySouthern blot hybridization with portions of human SR-BI cDNA, and shownto contain a large portion of the SR-BI sequence. This BAC was thensheared by nebulizing the DNA into fragments of approximately 1-3 kbwhich were inserted into the pminisk vector and the resulting insertsizes ranged from 1-3 kb. Initially, clones which hybridized to thecoding sequence of the full-length human SR-BI cDNA were sequenced,leading to the identification of most of the exons of the gene. Furtherrandom sequencing of the BAC sheared library led to the identificationof the remaining coding exons and the adjacent intron flankingsequences.

[0253] Sequence analysis of the genomic DNA indicated that the humanSR-BI gene is at least 50 kb and contains 12 coding exons and onenon-coding exon (exon 13, which contains the entire 3′ untranslatedregion). The genomic structure of human SR-BI is shown in FIG. 1. Thenucleotide sequence of the exons and portions of the introns which areadjacent to the exons is shown in FIG. 2A-G. The coding region of thehuman SR-BI gene consists of 12 exons (see Table IV in the DetailedDescription). The location of introns relative to the nucleotidesequence of a cDNA encoding human SR-BI is shown in FIG. 2A-G and FIG. 3and indicated in Table V of the Detailed Description. The portions ofthe protein encoded by each of the exons is also shown in FIG. 3 and inTable V.

[0254] A number of the introns are extremely large (>10 kb) (see TableIV in the Detailed Description). The intron/exon boundaries wereremarkably similar to those found in the human CD36 gene, which is amember of the same protein family as SR-BI (Tang et al. (1994) J. Biol.Chem. 269:6011).

Example 2

[0255] Identification of primer pairs to isolate intronic, exonic, andpromoter sequences for detection of polymorphisms and mutations

[0256] Multiple pairs of primers were synthesized in order to amplifyeach of the exons or portions thereof and adjacent intronic regions.Genomic DNA from a human subject was subjected to PCR in 25 μl reactions(1× PCR Amplitaq polymerase buffer, 0.1 mM dNTPs, 0.8 μM 5′ primer, 0.8μM 3′ primer, 0.75 units of Amplitaq polymerase, 50 ng genomic DNA)using each of the above described pairs of primers under the followingcycle conditions: 94° C. for 2 min, 35×[94° C. for 40 sec, annealingtemp for 30 sec, 72° C. for 1 min], 72° C. for 5 min, 4° C. hold. Theresulting PCR products were analyzed on a 2% agarose gel. The identityof the PCR product was confirmed by digestion with a restriction enzymeand subsequent agarose electrophoresis. Twelve pairs of oligomers werechosen to serve as PCR primers to amplify regions containing each of the12 coding exons of the human SR-BI gene and one pair of primers waschosen to serve as PCR primers to amplify a promoter region. Thenucleotide sequence of these primers in indicated in Table IX andnucleotide sequences to which these primers bind are shown in FIG. 2A-G.The optimum PCR annealing temperature for each primer pair as well asthe expected sizes of the PCR products and diagnostic restriction sitesis set forth below in Table VIII. Table VIII also indicates the size ofDNA fragments obtained when digesting the amplified fragment with therestriction enzyme indicated in the table. A PCR reaction using primershaving SEQ ID NO: 41 and 42 for amplifying exon 1 is preferably carriedout in the presence of 10% DMSO. TABLE VII primer SEQ NucleotideSequence exon name ID NO: (5′ −> 3′) 1 5e16srb1 41CCCCTGCCGCCGGAATCCTGAAG 3e16srb1 42 CGCTTTGGCGGAGCAGCCCATGTC 2 5e22srb143 TGGGGCCCTCATCACTCTCCTCAC 3e22srb1 44 GCAGCCTCCCCATCCCGTCCACT 35e30srb1 45 ATTGCAGGCGAGTAGAAG 3e30srb1 46 CAGGCGGGAGGAGAGACA 4 5e41srb147 TGGGCTCTTTGCTGTGAGGC 3e41srb1 48 CCAGGCTGTGTGAGGGGAAG 5 5e50srb1 49GCCCAGAATGTTCAGACCAG 3e50srb1 50 GCACCCTCTTCACGACAAAG 6 5e60srb1 51CACCTGAGAGGGCTTATTA 3e60srb1 52 CAAAATGCTTTCCAAGTGC 7 5e71srb1 53GCCGCCGGGTCTGGGTGTCC 3e71srb1 54 CAGAGGCCAGAGATTAAGCAGAC 8 5e81srb1 55TTGTATGATGTCCCCTCCCT 3e81srb1 56 TTCCCACCACCCCAGCCCAC 9 5e91srb1 57GGTTGACTGTGTCCCTGGAG 3e91srb1 58 GGGAACACTGGAGCACTGAGC 10 5e104srb1 59GGTGGTGAGGGTTTAGTGTG 3e104srb1 60 CTCCCCCCGCCTCCTGCCTC 11 5e112srb1 61AAGGTGTTGGGTGGCATCTG 3e112srb1 62 GGCTCCAGGCTGCGGTTGGC 12 5e100srb1 63TTGAAGAACCGTGTAAAAC 3e100srb1 64 TTGAGGCTGAAGGAATGA Prom. 5p13srb1 83TCCTGGGTGGGCTGGCGAAGTC 5p13srb1 84 GTTTTGGGGCGGGAGCTGATGAAG

[0257] TABLE VIII Exon primer pairs Temp. Product length Enzyme check 1SEQ ID NO: 41 65° C. 162 bp BamHI (144, 118) SEQ ID NO: 42 2 SEQ ID NO:43 64° C. 294 bp ApaI (189, 98, 7) SEQ ID NO: 44 3 SEQ ID NO: 45 57° C.281 bp XhoI (153, 128) SEQ ID NO: 46 4 SEQ ID NO: 47 59° C. 360 bp SpeI(292, 68) SEQ ID NO: 48 5 SEQ ID NO: 49 57° C. 291 bp BamHI (157, 134)SEQ ID NO: 50 6 SEQ ID NO: 51 52° C. 273 bp DraII (179, 72, 22) SEQ IDNO: 52 7 SEQ ID NO: 53 59° C. 290 bp EcoRI (184, 106) SEQ ID NO: 54 8SEQ ID NO: 55 58° C. 261 bp HaeIII(158, 103) SEQ ID NO: 56 9 SEQ ID NO:57 57° C. 206 bp PstI (107, 99) SEQ ID NO: 58 10  SEQ ID NO: 59 56° C.253 bp AvaII (148, 105) SEQ ID NO: 60 11  SEQ ID NO: 61 60° C. 327 bpNcoI (242, 85) SEQ ID NO: 62 12  SEQ ID NO: 63 51° C. 303 bp PstI(184,119) SEQ ID NO: 64 prom. SEQ ID NO: 83 63° C. 247 bp BstXI (200, 47) SEQID NO: 84

Example 3

[0258] Detection of polymorphic regions in the human SR-BI gene by SSCP

[0259] Genomic DNA from a population of 389 unrelated Caucasian men andwomen, chosen because they had a known HDL and LDL level (high, normal,or low), known body mass index, known level of triglycerides, and knownage (see Table IX) was analyzed as described below. TABLE IXAnthropometric and plasma lipid concentrations of the population studiedMen (n = 101) Women (n = 288) Age (years) 40 ± 16 36 ± 12 BMI (kg/m²)25.2 ± 3.3  22.8 ± 3.6  TC (mg/dL) 227 ± 57  198 ± 45  LDL-C (mg/dL) 158± 49  122 ± 39  HDL (mg/dL) 45 ± 23 63 ± 17 TG (mg/dL) 120 ± 64  68 ± 34

[0260] Genomic DNA from each of these individuals was subjected to PCRin 25 μl reactions (1X PCR Amplitaq polymerase buffer, 0.1 mM dNTPs, 0.8μM 5′ primer, 0.8 μM 3′ primer, 0.75 units of Amplitaq polymerase, 50 nggenomic DNA) using each of the above described pairs of primers underthe following cycle conditions: 94° C. for 2 min, 35×[94° C. for 40 sec,annealing temp for 30 sec, 72° C. for 1 min], 72° C. 5 min, 4° C. hold.The optimum PCR annealing temperatures for each set of primers are givenin Table VIII. The expected sizes of the PCR products, as well asdiagnostic restriction sites, are also indicated in Table VIII.

[0261] The amplified genomic DNA fragments were then analyzed by SSCP(Orita et al. (1989) PNAS USA 86:2766, see also Cotton (1993) Mutat Res285:125-144; and Hayashi (1992) Genet Anal Tech Appl 9:73-79). From each25 μl PCR reaction, 3 μl was taken and added to 7 μl of loading buffer.The mixture was heated to 94° C. for 5 min and then immediately cooledin a slurry of ice-water. 3-4 μl were then loaded on a 10%polyacrylamide gel containing 10% glycerol and then subjected toelectrophoresis either overnight at 4 Watts at room temperature,overnight at 4 Watts at 4° C. (for amplifying a promoter region), or for5 hours at 20 Watts at 4° C. (for amplifying exons 3 and 4). Thesecondary structure of single-stranded nucleic acids varies according tosequence, thus allowing the detection of small differences in nucleicacid sequence between similar nucleic acids. At the end of theelectrophoretic period, the DNA was analyzed by gently overlaying amixture of dyes onto the gel (1×the manufacturer's recommendedconcentration of SYBR Green I and SYBR Green II in 0.5×TBE buffer(Molecular Probes)) for 5 min, followed by rinsing in distilled waterand detection in a Fluoroimager 575 (Molecular Dynamics). Polymorphismswere found in or near exons 1, 3, 5, 8, and 10.

Example 4

[0262] Identificaion ofpolymorphic regions in the human SR-BI gene bydirect sequencing ofPCR products

[0263] To determine the sequences of the polymorphisms identified, theregions containing the polymorphisms were reamplified using theaforementioned primers which were modified to contain additionalsequence which could be used to directly sequence the PCR product (M13forward sequence for 5′ primer and +M13 reverse sequence for 3′ primer)on the 5′ end of the primers as listed in Table VII. In particular, theforward primers (5′ end primers) contained the nucleotide sequence“TGTAAAACGACGGCCAGT” (SEQ ID NO: 85) located 5′ of the nucleotidesequences shown in Table VII and the reverse primer (3′ end primer)contained the nucleotide sequence “CAGGAAACAGCTATGACC” (SEQ ID NO: 86)located 5′ of the nucleotide sequence shown in Table VI. The genomic DNAfrom the subjects was subjected to PCR in 50 μl reactions (1× PCRAmplitaq polymerase buffer, 0.1 mM dNTPs, 0.8 μM 5′ primer, 0.8 μM 3′primer, 0.75 units of Amplitaq polymerase, 50 ng genomic DNA) using eachof the above described pairs of primers under the following cycleconditions: 94° C. for 2 min, 35×[94° C. for 40 sec, annealing temp for30 sec, 72° C. for 1 min], 72° C. 5 min, 4° C. hold. The optimum PCRannealing temperatures for each of the primer pairs are given in TableVIII. The newly amplified products were then purified by agarose gelelectrophoresis and subjected to sequencing using M13 forward andreverse primers.

[0264] The results indicate that the polymorphism in the region of exon1 is a change from a guanine at nucleotide 146 to an adenine, resultingin a change of the second amino acid of the protein from a glycine to aserine. The polymorphism in the region of exon 3 is a change from theguanine at position to 119 to an adenine, resulting in a change of aminoacid 135 of the protein from a valine to isoleucine. The polymorphism inthe region of exon 8 was determined to constitute a change in baseposition 41 of exon 8, from a cytidine (referred to herein as EX8C) to athymidine (referred to herein as EX8T). This substitution does notresult in a change in amino acid. In a subpopulation of 142 individuals,about 35% of these individuals were homozygous for an allele having acytidine at position 41 of exon 8; about 17% of these individuals werehomozygous for an allele having a thymidine at this position; and about48% of these individuals were heterozygous, having one allele of eachtype. The polymorphism in the region of exon 5 is a change in nucleotide54 of intron 5 (nucleotide 1 being the first nucleotide of the intron)from a cytidine (referred to herein as IVS5C) to a thymidine (referredto herein as IVS5T). In a subpopulation of 142 individuals, about 24% ofindividuals have a thymidine at position 54 of intron 5. Thepolymorphism in the region of exon 11 is a change from the cytidine atposition −41 (nucleotide-1 corresponds to the most 3′ nucleotide ofintron 10) of intron 10 to a guanine. The polymorphisms are indicated inTable VI and in FIG. 2A-G.

Example 5

[0265] Association of common polymorphisms at the SR-BI gene with plasmalipids and anthropometric parameters

[0266] After identification of the mutations in exons 1, 3, and 8 andintrons 5 and 10, subjects were typed by digestion of PCR products usingthe primers and enzymes listed in Table X. TABLE X Primers and enzymesfor typing allelic variants poly- morphism primers temp digest productsizes exon 1 CCGGCGATGGGGCATAAAACCACT (SEQ ID NO.:89) 68-62 C. Alul GG:263 (G/A) CGCCCAGCACAGCGCACAGTAGC (SEQ ID NO.:90) GA: 263, 192, 71 AA:192, 71 intron 5 GCCCAGAATGTTCAGACCAG (SEQ ID NO.:91)    57 C. Apal CC:194, 67, 30 (C/T) GCACCCTCTTCACGACAAAG (SEQ ID NO:92) CT: 194, 97, 67,30 TT: 194, 97 exon 8 CCTTGTTTCTCTCCCATCCTCACTTCCTCAAGGC (SEQ ID NO.:93)66-61 C. Haelll CC: 154, 33, 31 (C/T) CACCACCCCAGCCCACAGCAGC (SEQ IDNO.:94) CT: 154, 64, 33, 31 TT: 154, 64

[0267] Plasma lipids were measured after a 12 to 14-hour overnight fast,using blood collected in tubes containing 0.1% EDTA. Plasma HDLcholesterol was measured after precipitation of plasma apoB-containinglipoproteins as previously described. Plasma total cholesterol, HDLcholesterol and triglyceride levels were measured as previouslydescribed. LDL cholesterol was calculated by the Friedewald equationwhen triglyceride levels were below 400 mg/dL. Coefficients of variationbetween runs for all lipid assays were less than 5%.

[0268] The SPSS statistical package PC version 7.5.1 was used for thestatistical analysis. Because of the differences observed between menand women for several of the anthropometric and lipid variables, all thestatistical analyses were carried out separately by gender.Triglycerides were log transformed for analysis. For each of thevariables examined, the significance of the differences between allelesor genotypes was estimated by analysis of covariance (ANCOVA) using theGeneral Linear Model (GLM) procedure from SPSS, with age as covariateand the Tukey's post hoc test for multiple comparisons for observedmeans. For analysis related to HDL, smoking and alcohol intake were alsoincluded as covariates. Means and standard deviations for all variableswere calculated for each genotype group and the significance level wasestablished at p<0.05. The allele and haplotype frequencies wereestimated using the EH linkage utility program (Terwilliger and Ott J.(1994) Handbook for human genetic linkage. Johns Hopkins UniversityPress, Baltimore, Md.).

[0269] The frequencies of the less common allele for each of thepolymorphisms described at the SR-BI gene locus were as follows: exon 1:0.1136; exon 3: 0.0184; intron 5: 0.1002; exon 8: 0.4389 and intron 11:0.0425. The associations between these common polymorphisms and plasmalipid concentrations and BMI are presented in table XI for men and tableXII for women. For men, no significant associations were observedbetween any of the variables examined and the common polymorphisms atexon 1, intron 5 and exon 8. Conversely, in women, the less commonallele defined by the polymorphism at exon 8 was associated withsignificantly lower mean plasma LDL cholesterol concentrations (118±38and 116±36 mg/dL for heterozygotes and homozygotes, respectively) thanthose observed in subjects homozygous for the most common allele (131±42mg/dL; p=0.043). No other significant associations were observed betweenthese polymorphisms and other lipid variables. In women, a significantassociation was observed between the intron 5 polymorphism and BMI.Women carriers of the less common allele showed a mean BMI value(23.8±3.8) that was significantly greater (p=0.031) than those womenhomozygous for the most common allele (22.4±3.4). TABLE XIAnthropometric characteristics and plasma lipid concentration of thepopulation studied according to SR-BI genotypes (Men) Exon 1 Intron 5Exon 8 1/1 1/2 P 1/1 1/2 P 1/1 1/2 2/2 P (n = 71) (n = 8) value (n = 74)(n = 19) value (n = 22) (n = 49) (n = 18) value Age  41 ± 14  38 ± 160.659  40 ± 15  46 ± 13 0.114  47 ± 14  42 ± 14  37 ± 13 0.068 (years)BMI 25.7 ± 3.0 25.6 ± 1.9 0.883 25.3 ± 2.6 26.1 ± 3.8 0.326 25.7 ± 3.625.8 ± 2.5 25.0 ± 3.2 0.583 (kg/m²) TC 228 ± 46 221 ± 38 0.712 225 ± 55240 ± 41 0.282 234 ± 40 221 ± 51 225 ± 47 0.559 (mg/dL) LDL-C 159 ± 46157 ± 47 0.908 156 ± 51 175 ± 48 0.145 168 ± 44 154 ± 52 158 ± 47 0.520(mg/dL) HDL  43 ± 22  43 ± 14 0.983  43 ± 24  45 ± 19 0.733  39 ± 17  43± 25  44 ± 16 0.761 (mg/dL) TG 127 ± 65 105 ± 43 0.346 129 ± 68  98 ± 270.052 133 ± 63 122 ± 60 119 ± 69 0.715 (mg/dL)

[0270] TABLE XII Anthropometric characteristics and plasma lipidconcentration of the population studied according to SR-BI genotypes(Women) Exon 1 Intron 5 Exon 8 1/1 1/2 2/2 P 1/1 1/2 P 1/1 1/2 2/2 P (n= 181) (n = 66) (n = 1) value (n = 74) (n = 19) value (n = 73) (n = 148)(n = 37) value Age  35 ± 12  38 ± 12 40 0.218  36 ± 12  38 ± 12 0.333 37 ± 12  36 ± 12  34 ± 12 0.458 (years) BMI 22.9 ± 3.9 22.5 ± 3.0 19.70.552 22.4 ± 3.4 23.8 ± 3.8 0.031 22.8 ± 3.0 23.0 ± 3.9 21.9 ± 3.4 0.252(kg/m²) TC 197 ± 45 198 ± 42 211 0.932 198 ± 46 204 ± 44 0.446 206 ± 49196 ± 42 192 ± 42 0.215 (mg/dL) LDL-C 121 ± 39 120 ± 38 134 0.928 122 ±39 125 ± 42 0.711 131 ± 42 118 ± 38 116 ± 36 0.043 (mg/dL) HDL  63 ± 17 64 ± 19 63 0.930  62 ± 16  64 ± 21 0.145  61 ± 16  64 ± 17  64 ± 220.500 (mg/dL) TG  64 ± 28  71 ± 37 72 0.317  68 ± 35  64 ± 30 0.423  67± 35  70 ± 38  57 ± 22 0.146 (mg/dL)

[0271] Haplatype analyses (using the method described by terwillingerand Ott, 1984, supra) using the three most common polymorphismsidentified at this locus (exon 1, intron 5 and exon 8) were carried out.Six possible haplotypes were identified according to the absence (1) orpresence (2) of the variant allele at each one of the three polymorphicsites. Four of the haplotypes were common: 111 (wild type); 112 (exon 8variant); 121 (intron 5 variant); 211 (exon 1 variant); whereas two ofthe estimated haplotypes were rare: 221 (variants at exon 1 and intron5) and 212 (variants at exons 1 and 8). The most common haplotype, 111,had the highest apparent frequency in women (44.6%) and therefore wasconsidered to be wild-type. Each subject was assigned to the mostplausible genotype; however, because of the uncertainty associated withgenotype assignments in double heterozygotes when studying unrelatedsubjects on whom the phase of the polymorphisms cannot be directlyascertained, we used in further analysis only those subjects withunequivocal genotypes. In women, the 111/112 and 112/112 genotypes werefound to be associated with lower LDL-C levels as compared with the wildgenotype (111/111); whereas the 111/121 genotype was associated withincreased LDL-C levels as compared with the 111/112 and 111/211genotypes (FIG. 4). In men the trends were similar to those observed inwomen; however, the differences did not reach statistical significance(FIG. 5). In terms of HDL levels, all the alleles carrying mutationswere associated with increased HDL levels in men (FIG. 7); however, nosignificant effects were observed in women (FIG. 6). Thus, as comparedto the wild-type haplotype, the analyses revealed that men withhaplotypes 112, 121, and 211 tended to have significantly higher HDL. Infact, those subjects with haplotype 211 (one wild-type chromosome andone with a polymorphism in exon 1) had an average of 75% higher HDLlevels than individuals containing only wild-type chromosomes. Suchassociations were not observed in women. Without wanting to be limitedby a specific mechanism of action, it has been shown that, though lessefficient than the LDL receptor, SR-BI is able to mediate thedegradation of LDL in vitro. SR-BI may also play an indirect role in LDLcholesterol metabolism by altering cholesterol homeostasis in theindividual.

[0272] The previous association between the intron 5 polymorphism andBMI observed in women using single marker analysis was stronger usinghaplotype analysis. Subjects carrying the 121 haplotype (n=12), had amean BMI (25.0″3.3) significantly greater (p<0.05) than that observed insubjects homozygous for the wild haplotype 111 (n=29; 22.8″3.2) andthose carriers of the 112 (n=78; 22.8″3.9) and 211 (n=12; 21.5″2.1)haplotypes (FIG. 8). However, no association was observed in men (FIG.9).

[0273] There was also a significant association between the intron 5polymorphism and BMI in women. The latter finding was most significantin premenopausal women. The 288 female subjects in this study were notconsidered to be obese. Thus, this effect was observed on individualswithin the normal weight range. There are only a few polymorphisms knownto date that are associated with BMI values in humans (Bouchard andPerusse (1996) Obesity Research 4:81-90), and thus this is a significantfinding. Increased body-mass index has been associated with highermortality from all causes and from cardiovascular disease. For mortalityfrom cardiovascular disease, the relative risk associated with anincrement of one in the body-mass index in women in the age range of30-to-44 year old has been reported to be 1.08 (95 percent confidenceinterval, 1.05 to 1.11) (Stevens et al. (1998) N Engl J Med 338:1-7;Colditz et al. (1995) Ann Intern Med. 122(7):481-486). The datapresented herein show that for an average woman, the presence of the 121haplotype raises the BMI by approximately 2.2 kg/m2 which corresponds toabout 6 kgs. This increase in BMI could result in an increase in CHDmortality of about 17.6%, primarily due to a greater risk of developingnon-insulin dependent diabetes mellitus (Stevens et al. (1998) supra;Colditz et al. (1995) supra), a major risk for coronary arteryatherosclerosis. An intriguing observation is the fact that no subjectswere found to be homozygous for this polymorphism in this sample of 389randomly selected individuals, despite the expected frequency of 1% (˜4individuals) assuming Hardy-Weinberg equilibrium.

Example 6

[0274] Association of common polymorphisms at the SR-BI gene with plasmalipids in three ethnically distinct populations

[0275] Study populations

[0276] Subjects were drawn from three collections of nuclear familiesascertained for T2DM. Two Scandinavian populations were recruited: onefrom Finland and one from southern Sweden. The third was a population ofIsraelis of Ashkenazi Jewish origin. Nuclear families were identifiedhaving at least two members with T2DM. Those patients with a diagnosisbefore the age of 70 were invited to participate. In the Ashkenazistudy, at least one sibling had to have initial diagnosis prior to age56. All subjects with age of onset <35 years were excluded to minimizethe inclusion of Maturity Onset of the Young (MODY) and Type 1 diabetesmellitus. Furthermore, patients who became insulin-dependent within twoyears of diagnosis were excluded. To avoid bilinial inheritance,families in which both parents were known to have diabetes wereexcluded. T2DM was diagnosed using WHO criteria (World HealthOrganization Study Group Diabetes Mellitus (1985) Technical reportseries No. 727, WHO, Geneva), e.g., fasting blood glucose >6.7 mmol/l ortwo hour blood glucose ≧10.0 mmol/l. Individuals lacking fasting bloodglucose and oral glucose tolerance test data were considered affected ifcurrently taking oral hypoglycemics and/or insulin. To test unaffectedstatus, all presumed unaffected siblings were asked to undergo astandard oral glucose tolerance test, however, inclusion in the studywas not dependent on their agreement to undergo this test. These studieswere approved by the local Institutional Review Boards in Israel,Finland and Sweden. Informed consent was obtained from all participants.

[0277] Blood samples were obtained following an overnight fast of atleast 12 hours by drawing into tubes containing EDTA. Plasma HDL, totalcholesterol and TG were measured using standard procedures. Clinicaldata, including anthropometric measures such as body mass index (BMI;weight in kilograms/height in meters squared), were gathered for eachsubject on the day of enrollment.

[0278] SRBI variants

[0279] The SR-B1 variants evaluated were chosen based on allelefrequencies to maximize the ability to detect an association. The mostcommon DNA variants described in SR-B1 are a silent single nucleotidepolymorphism (SNP) in exon 8 at amino acid 350, and a SNP 54 nucleotidesinto intron 5 (IVS5+54C→T). The SR-B1 variants in exon 8 (EX8; allelesEX8C. and EX8T) and intron 5 (IVS5; alleles IVS5C and IVS5T) wereexamined to determine if these two SNPs alone or in combination wereassociated with HDL levels.

[0280] Genotyping Genomic DNA was isolated from peripheral bloodlymphocytes using the Puregene kit (Gentra Systems, Inc.) according tomanufacturer's suggested protocol. The IVS5 and EX8 polymorphisms wereamplified under the following PCR conditions: 2.5 mM MgCl2, 1× AmmoniumSulfate buffer (80 mM (NH4)2SO4, 335 mM Tris-HCl pH 8.8, 0.05% Tween20), 200 μM dNTP, 0.015 U Platinum Taq polymerase (Life Technologies,Inc.), 0.17 μM each of forward and reverse primers, and 5 ng genomic DNAin a 15 μL reaction volume. Forward PCR primers, shown in Table X, werefluorescently labeled at the 5′ end. Thermocycling conditions were 95°C. for 10 min, 95° C. for 30 s, 55° C. for 30 s, 72° C. for 1 min, for35 cycles. Variants were visualized by Single-Stranded ConformationPolymorphism (SSCP) on ABI Prism 377 DNA Sequencers (Applied BiosystemsGroup) by loading products on a non-denaturing 7% Long Ranger acrylamidegel (BioWittiker Molecular Applications) at 75 W, 15° C., for 10 h.TABLE XIII Sequence of primers used to genotype SR-B1 variants in intron5 and exon 8. Product Location on Polymorphism Primer sequence size (bp)SSCP gel Allele IVS5 (C/T) TCACGGGGGTCCAGAACATC (SEQ ID NO:122) 174upper band T TTCACGACAAAGGAAGAAGGAGC (SEQ ID NO:123) lower band C EX8(C/T) TGTCGGGTATTATGGTCATCGCC (SEQ ID NO:124) 238 upper band TATGTCCACGAACAAGGAGTGTGC (SEQ ID NO:125) lower band C

[0281] Design of nested case-control studies

[0282] Sex-specific thresholds were used for defining low HDL cases andhigh HDL controls. Cases and controls were chosen without respect toT2DM status. For women, low HDL was defined as having HDL≦1.16 mmol/l(45 mg/dl), representing the median HDL level in women from all threepopulations combined. Female controls had HDL >1.16 mmol/l. For men, lowHDL was defined as having HDL≦1.01 mmol/l (39 mg/dl), representing themedian HDL level in men from all three populations combined. Malecontrols had HDL>1.01 mmol/l.

[0283] Inclusion of multiple family members in either case series orcontrol series may affect the distribution of genotypes and lead to anoverestimate of the significance of the association. Therefore, for eachsex an independent case series and independent control series wasdefined. If more than one case existed in the same family, the case withthe younger age was selected to remain in the case series. If more thanone control existed from the same family, the control with the youngerage was selected to remain in the control series. Younger aged controlswere chosen to improve matching on age-related covariates. If both acase and control were found in the same family, the case was used andthe control eliminated.

[0284] Statistical analysis

[0285] All analyses were performed using the SAS statistical packageversion 6.12 (SAS Institute Inc., Cary, N.C.). Differences in meanlevels of HDL between genotype groups was compared using one-wayanalysis of variance (ANOVA). A T-Test was used to assess differencesbetween cases and controls with respect to age, BMI and TG. Since TGlevels had a skewed distribution, the statistical analyses were based onlog-transformed data. However, in the tables the TG levels are given asmean (±SD). The chi square statistic was used to compare the proportionof T2DM among cases and controls. Odds ratios and 95% confidenceintervals were calculated for contingency tables and the overallsignificance assessed using the continuity adjusted chi square. Logisticregression analysis was performed using the PROC LOGISTIC procedure inSAS. BMI, age and TG were entered into the models as continuousvariables while T2DM status was dichotomous. Linkage disequilibrium wasmeasured using the normalized disequilibrium parameter (Lewontin, R C(1964) Genetics 49:49-67), D′, and the statistical significance assessedwith a chi square test. Relative risk and attributable risk estimateswere calculated as described below.

[0286] Calculation of relative risk and attributable risk fromcase-control data.

[0287] RR=relative risk; OR=odds ratio; D=outcome; G=risk genotype.

[0288] In this study, p(D), the frequency of low HDL, is ˜50% as it wasdefined as being below the population median.

[0289] The following conditional probabilities can be estimated from thetwo by two table of case-control data: p(G|D) p(G|noD) p(noG|D)p(noG|noD)

[0290] Since p(D) is known, P(G) can be calculated using the law oftotal probability:

P(G)=p(G|D)p(D)+p(G|noD)p(noD)

[0291] Relative risk and odds ratio estimates are related in thefollowing way:

RR=R₁/R₀ OR=RR(1-R₀)/(1-R₁) RR=OR(1-R₁)/(1-R₀)

[0292] R₁ and R₀ can be expressed as conditional probabilities: R₁ =p(D|G) R₀ = p(D|noG) (1-R₁) = p(noD|G) (1-R₀) = p(noD|noG)

[0293] Using Bayes theorem, these conditional probabilities can beexpressed as:

p(noD|G)=p(G|noD) p(noD)/p(G) p(noD|noG)=p(noG|noD) p(noD)/p(noG)

[0294] Therefore: (1-R₁)/(1-R₀)=[p(G|noD) p(noD)/p(G)]/[p(noG|noD)p(noD)/p(noG)] and relative risk can be estimated as:

RR=OR[p(noG) p(G|noD)/p(G) p(noG|noD)]

[0295] Attributable risk is calculated from the following formuladescribed by Coughlin et al²⁶.${AR} = {1 - \frac{\sum( {n_{j}/R_{j}} )}{N}}$

[0296] where j are the strata defined by presence or absence of thegenotype, n_(j) is the number of cases in each stratum and R_(j) is therelative risk associated with each stratum. N is the total number ofcases.

[0297] Results

[0298] Mean HDL levels were correlated with SR-B1 EX8 genotype for allfamily members with genotype data available (1649 individualsrepresenting 600 families from three populations). All three populationssuggested an association between SR-B1 genotype and HDL level.Specifically, carriers of EX8C. have lower HDL than non-carriers. Two ofthe populations, the Ashkenazi and Finnish, showed marked differences inthis effect by sex with the effect being more pronounced in women. Tofurther characterize the association of SR-B1 variants with HDL, nestedsex-specific case control studies were designed for each population andsex. Table XIV, below, shows the breakdown of cases and controls andtheir sampling from the initial collection of families. The final samplesize included 558 low HDL cases and 379 high HDL controls. Eachpopulation and sex was examined separately to determine the associationof SR-B1 EX8 and IVS5 genotypes with low HDL. TABLE XIV Breakdown ofindividuals chosen for nested case-control studies. WOMEN MEN AKZ FINSWE AKZ FIN SWE all individuals genotyped: 318 346 221 266 273 225 allcases (low HDL): 171 162 89 152 110 100 all controls (high HDL): 147 184132 114 163 125 independent cases: 121 108 65 113 78 73 independentcontrols: 114 110 89 87 114 92 case & control in same 32 61 33 23 42 46family: final cases 121 108 65 113 78 73 final controls: 82 49 56 64 7256

[0299] Characteristics of the six population and sex-specific pairs ofcases and controls are shown in table XV, below. The average age ofstudy participants varied between 56 and 63 years/Significantdifferences in age between cases and controls were found for Ashkenaziwomen (p<0.0006) and Swedish men (p<0.01). Because the cases andcontrols were chosen from families originally ascertained for T2DM, theyare enriched for T2DM and related phenotyhpes. Most (80%) of cases andcontrols combined had T2DM. However, significant differences in thefrequency of T2DM between cases and controls were found only for finnishwomen and men (both p<0.01). Significant differences in mean BMI betweencases and controls were found only for Swedish women (p<0.01). Allpopulations were enriched for low HDL and high TG, the hallmarkdyslipidemia associated with T2DM. HDL and TG levels are closely linkedand reflected in the fact that all six pairs of cases and controlsdefined as such based on HDL levels, also differed significantly intheir levels of TG. TABLE XV Characteristics of cases and controls bysex and population. WOMEN MEN low HDL high HDL low HDL high HDLASHKENAZI No. individuals 121 82 113 64 % T2DM 75% 85% 82% 91% mean age57.9 ± 11.5 63.0 ± 9.4* 57.7 ± 12.3 59.8 ± 10.2 mean BMI 28.7 ± 4.5 28.4± 4.8 27.3 ± 3.7 27.4 ± 4.0 mean HDL 0.89 ± .21 1.46 ± .27† 0.76 ± .191.28 ± .34† (mmol/l) mean TG 2.37 ± 1.41 1.71 ± 76* 2.41 ± 1.87 1.69 ±1.00* (mmol/l) FINNISH No. individuals 108 49 78 72 % T2DM 84% 59% 72%90%‡ mean age 59.7 ± 11.9 58.4 ± 13.1 56.4 ± 11.9 57.5 ± 11.5 mean BMI30.5 ± 5.2 28.5 ± 5.8 31.8 ± 17.1 27.9 ± 3.9 mean HDL 1.00 ± .13 1.50 ±.23† 0.87 ± .10 1.32 ± .32† (mmol/l) mean TG 1.97 ± .82 1.26 ± .64† 2.19± 1.08 1.42 ± .76† (mmol/l) SWEDISH No. individuals 65 56 73 56 % T2DM82% 73% 79% 79% mean age 58.4 ± 13.4 58.2 ± 13.0 56.1 ± 13.5 61.4 ± 9.8‡mean BMI 29.4 ± 5.6 26.2 ± 4.6‡ 27.8 ± 3.9 26.3 ± 4.0 mean HDL 0.93 ±.15 1.51 ± .29† 0.85 ± .12 1.31 ± .24† (mmol/l) mean TG 2.14 ± 1.06 1.36± .67† 2.40 ± 1.82 1.39 ± .62† (mmol/l)

[0300] The association of each of the two SR-B1 variants with HDL wasassessed separately in both univariate and multivariate analyses.Unadjusted and adjusted odds ratios, taking into account T2DM, age, BMIand TG levels, for the association of SR-B1 EX8 and IVS5 genotypes withlow HDL are presented in Table I for women and Table II for men, in theDetailed Description. Univariate analyses demonstrated consistent andstrong associations between presence of EX8C. and low HDL in women fromall three ethnic groups. Unadjusted odds ratios were 2.59, 2.92 and 2.33for the Ashkenazi, Finnish and Swedish women, respectively. Allpopulations achieved statistical significance although the Swedishpopulation was borderline (p=0.054). In men, carriers of EX8C. alsodemonstrated increased odds of having low HDL, although the effect wasnot as strong as in women. Unadjusted odds ratios were 1.95, 1.38 and2.82 for the Ashkenazi, Finnish and Swedish populations, respectively.Only the Finnish population failed to reach statistical significance(p=.50). Odds ratios are only estimates of the underlying relative riskand should therefore be interpreted cautiously. Whereas the combinedpopulation unadjusted odds ratios for EX8C. carriers were 2.66(p<0.0000 1) for women and 1.81 (p=0.008) for men, the relative risks(calculated using the formulas set forth below) are more modest; 1.74for women and 1.40 for men.

[0301] Odds ratios for the association between carriers of IVS5T and lowHDL were consistently elevated across all three ethnic groups; 2.06,4.95 and 3.34 for Ashkenazi, Finnish and Swedish women, respectively.Only the Swedish reached statistical significance (p=0.013). For men,presence of IVS5T had the opposite effect, being inversely associatedwith low HDL. Odds ratios were 0.97, 0.37 and 0.30 for Ashkenazi,Finnish and Swedish men, respectively. Only the Swedish reachedstatistical significance (p=0.024).

[0302] Multivariate logistic regression analysis was performed tocontrol for the possible confounding effects of T2DM, age, BMI and TG onthe association of SR-B1 genotypes with HDL. Inclusion of these terms ascovariates in the logistic regression models had no marked effect on theassociation between SR-B1 variants and low HDL. Adjusted odds ratios arepresented in Tables I and II in the Detailed Description. SR-B1genotypes remained as strong, independent predictors of low HDL. Anadditional logistic regression model was run to test for the interactionof ethnic group with SR-B1 exon 8 genotype. It revealed no significantinteraction in either men or women. Therefore data on all three ethnicgroups were combined. Adjusted odds ratios for the association of SR-B1genotypes with HDL in the combined populations were 2.84 (p<0.0001) and2.32 (p=0.01) in women for EX8C. and IVS5T, respectively. In men, theadjusted odds ratios were 1.79 (p=0.015) and 0.64 (p=.15) for EX8C. andIVS5T, respectively.

[0303] Both EX8C. and IVS5T alone were positively associated with lowHDL in women. In men, EX8C. appears to have a positive association withlow HDL while IVS5T shows an inverse, borderline significantassociation. EX8C. and IVS5T are in complete positive linkagedisequilibrium in the Ashkenazi and Finnish populations (D′=1.0, p<10⁻⁶)and near complete linkage disequilibrium in the Swedish population(D′=.91, p<3×10⁻⁶). The combined effect of the two variants on low HDLare shown in Table III. In the Detailed Description. Among women,carriers of both variants had the highest odds of having low HDL(OR=4.79, p<0.00001). However, even carriers of EX8C. alone were atincreased odds of having low HDL relative to those without eithervariant (OR=2.44, p<0.0001).

[0304] The combination of variants at EX8 and IVS5 yielded somewhatdifferent results in men. As in women, male carriers of EX8C. who lackedIVS5T were at increased odds of having low HDL (OR=1.95, p<0.01).However, unlike women, male carriers of both EX8C. and IVS5T were nomore likely to have low HDL than individuals with neither variant(OR=1.08, p=.95). This difference was not due to population (ethnic)differences, as consistent associations are noted across the ethnicgroups.

[0305] Attributable risk estimates were calculated from the data for menand women separately to determine the portion of low HDL which may beattributable to having the SR-B1 EX8. By definition, p(D), the frequencyof low HDL in families of T2DM patients, was about 50% since the cutoffsfor choosing cases and controls were based on population medians. Usingthe formulas presented above (see “Calculation of relative risk andattributable risk from case-control data”), risk of low HDL attributableto carrying SR-B1 EX8C. was calculated to be 35% for women and 23% formen in this population.

[0306] All of the above-cited references, patents and publications arehereby incorporated by reference.

[0307] Equivalents

[0308] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

1 121 1 2630 DNA Human CDS (119)..(1645) 1 accgtgcctc tgcggcctgcgtgcccggag tccccgcctg tgtcgtctct gtcgccgtcc 60 ccgtctcctg ccaggcgcggagccctgcga gccgcgggtg ggccccaggc gcgcagac 118 atg ggc tgc tcc gcc aaagcg cgc tgg gct gcc ggg gcg ctg ggc gtc 166 Met Gly Cys Ser Ala Lys AlaArg Trp Ala Ala Gly Ala Leu Gly Val 1 5 10 15 gcg ggg cta ctg tgc gctgtg ctg ggc gct gtc atg atc gtg atg gtg 214 Ala Gly Leu Leu Cys Ala ValLeu Gly Ala Val Met Ile Val Met Val 20 25 30 ccg tcg ctc atc aag cag caggtc ctt aag aac gtg cgc atc gac ccc 262 Pro Ser Leu Ile Lys Gln Gln ValLeu Lys Asn Val Arg Ile Asp Pro 35 40 45 agt agc ctg tcc ttc aac atg tggaag gag atc cct atc ccc ttc tat 310 Ser Ser Leu Ser Phe Asn Met Trp LysGlu Ile Pro Ile Pro Phe Tyr 50 55 60 ctc tcc gtc tac ttc ttt gac gtc atgaac ccc agc gag atc ctg aag 358 Leu Ser Val Tyr Phe Phe Asp Val Met AsnPro Ser Glu Ile Leu Lys 65 70 75 80 ggc gag aag ccg cag gtg cgg gag cgcggg ccc tac gtg tac agg gag 406 Gly Glu Lys Pro Gln Val Arg Glu Arg GlyPro Tyr Val Tyr Arg Glu 85 90 95 ttc agg cac aaa agc aac atc acc ttc aacaac aac gac acc gtg tcc 454 Phe Arg His Lys Ser Asn Ile Thr Phe Asn AsnAsn Asp Thr Val Ser 100 105 110 ttc ctc gag tac cgc acc ttc cag ttc cagccc tcc aag tcc cac ggc 502 Phe Leu Glu Tyr Arg Thr Phe Gln Phe Gln ProSer Lys Ser His Gly 115 120 125 tcg gag agc gac tac atc gtc atg ccc aacatc ctg gtc ttg ggt gcg 550 Ser Glu Ser Asp Tyr Ile Val Met Pro Asn IleLeu Val Leu Gly Ala 130 135 140 gcg gtg atg atg gag aat aag ccc atg accctg aag ctc atc atg acc 598 Ala Val Met Met Glu Asn Lys Pro Met Thr LeuLys Leu Ile Met Thr 145 150 155 160 ttg gca ttc acc acc ctc ggc gaa cgtgcc ttc atg aac cgc act gtg 646 Leu Ala Phe Thr Thr Leu Gly Glu Arg AlaPhe Met Asn Arg Thr Val 165 170 175 ggt gag atc atg tgg ggc tac aag gacccc ctt gtg aat ctc atc aac 694 Gly Glu Ile Met Trp Gly Tyr Lys Asp ProLeu Val Asn Leu Ile Asn 180 185 190 aag tac ttt cca ggc atg ttc ccc ttcaag gac aag ttc gga tta ttt 742 Lys Tyr Phe Pro Gly Met Phe Pro Phe LysAsp Lys Phe Gly Leu Phe 195 200 205 gct gag ctc aac aac tcc gac tct gggctc ttc acg gtg ttc acg ggg 790 Ala Glu Leu Asn Asn Ser Asp Ser Gly LeuPhe Thr Val Phe Thr Gly 210 215 220 gtc cag aac atc agc agg atc cac ctcgtg gac aag tgg aac ggg ctg 838 Val Gln Asn Ile Ser Arg Ile His Leu ValAsp Lys Trp Asn Gly Leu 225 230 235 240 agc aag gtt gac ttc tgg cat tccgat cag tgc aac atg atc aat gga 886 Ser Lys Val Asp Phe Trp His Ser AspGln Cys Asn Met Ile Asn Gly 245 250 255 act tct ggg caa atg tgg ccg cccttc atg act cct gag tcc tcg ctg 934 Thr Ser Gly Gln Met Trp Pro Pro PheMet Thr Pro Glu Ser Ser Leu 260 265 270 gag ttc tac agc ccg gag gcc tgccga tcc atg aag cta atg tac aag 982 Glu Phe Tyr Ser Pro Glu Ala Cys ArgSer Met Lys Leu Met Tyr Lys 275 280 285 gag tca ggg gtg ttt gaa ggc atcccc acc tat cgc ttc gtg gct ccc 1030 Glu Ser Gly Val Phe Glu Gly Ile ProThr Tyr Arg Phe Val Ala Pro 290 295 300 aaa acc ctg ttt gcc aac ggg tccatc tac cca ccc aac gaa ggc ttc 1078 Lys Thr Leu Phe Ala Asn Gly Ser IleTyr Pro Pro Asn Glu Gly Phe 305 310 315 320 tgc ccg tgc ctg gag tct ggaatt cag aac gtc agc acc tgc agg ttc 1126 Cys Pro Cys Leu Glu Ser Gly IleGln Asn Val Ser Thr Cys Arg Phe 325 330 335 agt gcc ccc ttg ttt ctc tcccat cct cac ttc ctc aac gcc gac ccg 1174 Ser Ala Pro Leu Phe Leu Ser HisPro His Phe Leu Asn Ala Asp Pro 340 345 350 gtt ctg gca gaa gcg gtg actggc ctg cac cct aac cag gag gca cac 1222 Val Leu Ala Glu Ala Val Thr GlyLeu His Pro Asn Gln Glu Ala His 355 360 365 tcc ttg ttc ctg gac atc cacccg gtc acg gga atc ccc atg aac tgc 1270 Ser Leu Phe Leu Asp Ile His ProVal Thr Gly Ile Pro Met Asn Cys 370 375 380 tct gtg aaa ctg cag ctg agcctc tac atg aaa tct gtc gca ggc att 1318 Ser Val Lys Leu Gln Leu Ser LeuTyr Met Lys Ser Val Ala Gly Ile 385 390 395 400 gga caa act ggg aag attgag cct gtg gtc ctg ccg ctg ctc tgg ttt 1366 Gly Gln Thr Gly Lys Ile GluPro Val Val Leu Pro Leu Leu Trp Phe 405 410 415 gca gag agc ggg gcc atggag ggg gag act ctt cac aca ttc tac act 1414 Ala Glu Ser Gly Ala Met GluGly Glu Thr Leu His Thr Phe Tyr Thr 420 425 430 cag ctg gtg ttg atg cccaag gtg atg cac tat gcc cag tac gtc ctc 1462 Gln Leu Val Leu Met Pro LysVal Met His Tyr Ala Gln Tyr Val Leu 435 440 445 ctg gcg ctg ggc tgc gtcctg ctg ctg gtc cct gtc atc tgc caa atc 1510 Leu Ala Leu Gly Cys Val LeuLeu Leu Val Pro Val Ile Cys Gln Ile 450 455 460 cgg agc caa gag aaa tgctat tta ttt tgg agt agt agt aaa aag ggc 1558 Arg Ser Gln Glu Lys Cys TyrLeu Phe Trp Ser Ser Ser Lys Lys Gly 465 470 475 480 tca aag gat aag gaggcc att cag gcc tat tct gaa tcc ctg atg aca 1606 Ser Lys Asp Lys Glu AlaIle Gln Ala Tyr Ser Glu Ser Leu Met Thr 485 490 495 tca gct ccc aag ggctct gtg ctg cag gaa gca aaa ctg tagggtcctg 1655 Ser Ala Pro Lys Gly SerVal Leu Gln Glu Ala Lys Leu 500 505 aggacaccgt gagccagcca ggcctggccgctgggcctga ccggcccccc agcccctaca 1715 ccccgcttct cccggactct cccagcagacagccccccag ccccacagcc tgagcctccc 1775 agctgccatg tgcctgttgc acacctgcacacacgccctg gcacacatac acacatgcgt 1835 gcaggcttgt gcagacactc agggatggagctgctgctga agggacttgt agggagaggc 1895 tcgtcaacaa gcactgttct ggaaccttctctccacgtgg cccacaggcc tgaccacagg 1955 ggctgtgggt cctgcgtccc cttcctcgggtgagcctggc ctgtcccgtt cagccgttgg 2015 gcccaggctt cctcccctcc aaggtgaaacactgcagtcc cggtgtggtg gctccccatg 2075 caggacgggc caggctggga gtgccgccttcctgtgccaa attcagtggg gactcagtgc 2135 ccaggccctg gccacgagct ttggccttggtctacctgcc aggccaggca aagcgccttt 2195 acacaggcct cggaaaacaa tggagtgagcacaagatgcc ctgtgcagct gcccgagggt 2255 ctccgcccac cccggccgga ctttgatccccccgaagtct tcacaggcac tgcatcgggt 2315 tgtctggcgc ccttttcctc cagcctaaactgacatcatc ctatggactg agccggccac 2375 tytytggccg aagtggccgc aggctgtgcccccgagctgc ccccaccccc tcacagggtc 2435 cctcagatta taggtgccca ggctgaggtgaagaggcctg ggggccctgc cttccgggcg 2495 ctcctggacc ctggggcaaa cctgtgacccttttctactg gaatagaaat gagttttatc 2555 atctttgaaa aataattcac tcttgaagtaataaacgttt aaaaaaatgg gaaaaaaaaa 2615 aaaaaaaaaa aaaaa 2630 2 509 PRTHuman 2 Met Gly Cys Ser Ala Lys Ala Arg Trp Ala Ala Gly Ala Leu Gly Val1 5 10 15 Ala Gly Leu Leu Cys Ala Val Leu Gly Ala Val Met Ile Val MetVal 20 25 30 Pro Ser Leu Ile Lys Gln Gln Val Leu Lys Asn Val Arg Ile AspPro 35 40 45 Ser Ser Leu Ser Phe Asn Met Trp Lys Glu Ile Pro Ile Pro PheTyr 50 55 60 Leu Ser Val Tyr Phe Phe Asp Val Met Asn Pro Ser Glu Ile LeuLys 65 70 75 80 Gly Glu Lys Pro Gln Val Arg Glu Arg Gly Pro Tyr Val TyrArg Glu 85 90 95 Phe Arg His Lys Ser Asn Ile Thr Phe Asn Asn Asn Asp ThrVal Ser 100 105 110 Phe Leu Glu Tyr Arg Thr Phe Gln Phe Gln Pro Ser LysSer His Gly 115 120 125 Ser Glu Ser Asp Tyr Ile Val Met Pro Asn Ile LeuVal Leu Gly Ala 130 135 140 Ala Val Met Met Glu Asn Lys Pro Met Thr LeuLys Leu Ile Met Thr 145 150 155 160 Leu Ala Phe Thr Thr Leu Gly Glu ArgAla Phe Met Asn Arg Thr Val 165 170 175 Gly Glu Ile Met Trp Gly Tyr LysAsp Pro Leu Val Asn Leu Ile Asn 180 185 190 Lys Tyr Phe Pro Gly Met PhePro Phe Lys Asp Lys Phe Gly Leu Phe 195 200 205 Ala Glu Leu Asn Asn SerAsp Ser Gly Leu Phe Thr Val Phe Thr Gly 210 215 220 Val Gln Asn Ile SerArg Ile His Leu Val Asp Lys Trp Asn Gly Leu 225 230 235 240 Ser Lys ValAsp Phe Trp His Ser Asp Gln Cys Asn Met Ile Asn Gly 245 250 255 Thr SerGly Gln Met Trp Pro Pro Phe Met Thr Pro Glu Ser Ser Leu 260 265 270 GluPhe Tyr Ser Pro Glu Ala Cys Arg Ser Met Lys Leu Met Tyr Lys 275 280 285Glu Ser Gly Val Phe Glu Gly Ile Pro Thr Tyr Arg Phe Val Ala Pro 290 295300 Lys Thr Leu Phe Ala Asn Gly Ser Ile Tyr Pro Pro Asn Glu Gly Phe 305310 315 320 Cys Pro Cys Leu Glu Ser Gly Ile Gln Asn Val Ser Thr Cys ArgPhe 325 330 335 Ser Ala Pro Leu Phe Leu Ser His Pro His Phe Leu Asn AlaAsp Pro 340 345 350 Val Leu Ala Glu Ala Val Thr Gly Leu His Pro Asn GlnGlu Ala His 355 360 365 Ser Leu Phe Leu Asp Ile His Pro Val Thr Gly IlePro Met Asn Cys 370 375 380 Ser Val Lys Leu Gln Leu Ser Leu Tyr Met LysSer Val Ala Gly Ile 385 390 395 400 Gly Gln Thr Gly Lys Ile Glu Pro ValVal Leu Pro Leu Leu Trp Phe 405 410 415 Ala Glu Ser Gly Ala Met Glu GlyGlu Thr Leu His Thr Phe Tyr Thr 420 425 430 Gln Leu Val Leu Met Pro LysVal Met His Tyr Ala Gln Tyr Val Leu 435 440 445 Leu Ala Leu Gly Cys ValLeu Leu Leu Val Pro Val Ile Cys Gln Ile 450 455 460 Arg Ser Gln Glu LysCys Tyr Leu Phe Trp Ser Ser Ser Lys Lys Gly 465 470 475 480 Ser Lys AspLys Glu Ala Ile Gln Ala Tyr Ser Glu Ser Leu Met Thr 485 490 495 Ser AlaPro Lys Gly Ser Val Leu Gln Glu Ala Lys Leu 500 505 3 1825 DNA Human CDS(156)..(1682) 3 gccacctgca gggctactgc tgctccggcc actgcctgag actcaccttgctggaacgtg 60 agcctcggct tctgtcatct ctgtggcctc tgtcgcttct gtcgctgtcccccttcagtc 120 cctgagcccc gcgagcccgg gccgcacacg cggac atg ggc ggc agcgcc agg 173 Met Gly Gly Ser Ala Arg 1 5 gcg cgc tgg gtg gcg gtg ggg ctgggc gtc gtg ggg ctg ctg tgc gct 221 Ala Arg Trp Val Ala Val Gly Leu GlyVal Val Gly Leu Leu Cys Ala 10 15 20 gtg ctc ggt gtg gtt atg atc ctc gtgatg ccc tcg ctc atc aaa cag 269 Val Leu Gly Val Val Met Ile Leu Val MetPro Ser Leu Ile Lys Gln 25 30 35 cag gta ctg aag aat gtc cgc ata gac cccagc agc ctg tcc ttt gca 317 Gln Val Leu Lys Asn Val Arg Ile Asp Pro SerSer Leu Ser Phe Ala 40 45 50 atg tgg aag gag atc cct gta ccc ttc tac ttgtcc gtc tac ttc ttc 365 Met Trp Lys Glu Ile Pro Val Pro Phe Tyr Leu SerVal Tyr Phe Phe 55 60 65 70 gag gtg gtc aat ccc agc gag atc cta aag ggtgag aag cca gta gtg 413 Glu Val Val Asn Pro Ser Glu Ile Leu Lys Gly GluLys Pro Val Val 75 80 85 cgg gag cgt gga ccc tat gtc tac agg gaa ttc agacat aag gcc aac 461 Arg Glu Arg Gly Pro Tyr Val Tyr Arg Glu Phe Arg HisLys Ala Asn 90 95 100 atc acc ttc aat gac aat gat act gtg tcc ttt gtggag cac cgc agc 509 Ile Thr Phe Asn Asp Asn Asp Thr Val Ser Phe Val GluHis Arg Ser 105 110 115 ctc cat ttc cag ccg gac agg tcc cac ggc tct gagagt gac tac att 557 Leu His Phe Gln Pro Asp Arg Ser His Gly Ser Glu SerAsp Tyr Ile 120 125 130 ata ctg cct aac att ctg gtc ttg ggg ggc gca gtaatg atg gag agc 605 Ile Leu Pro Asn Ile Leu Val Leu Gly Gly Ala Val MetMet Glu Ser 135 140 145 150 aag tct gca ggc ctg aag ctg atg atg acc ttgggg ctg gcc acc ttg 653 Lys Ser Ala Gly Leu Lys Leu Met Met Thr Leu GlyLeu Ala Thr Leu 155 160 165 ggc cag cgt gcc ttt atg aac cga aca gtt ggtgag atc ctg tgg ggc 701 Gly Gln Arg Ala Phe Met Asn Arg Thr Val Gly GluIle Leu Trp Gly 170 175 180 tat gag gat ccc ttc gtg aat ttt atc aac aaatac tta cca gac atg 749 Tyr Glu Asp Pro Phe Val Asn Phe Ile Asn Lys TyrLeu Pro Asp Met 185 190 195 ttc ccc atc aag ggc aag ttc ggc ctg ttt gttgag atg aac aac tca 797 Phe Pro Ile Lys Gly Lys Phe Gly Leu Phe Val GluMet Asn Asn Ser 200 205 210 gac tct ggg ctc ttc act gtg ttc acg ggc gtccag aac ttc agc aag 845 Asp Ser Gly Leu Phe Thr Val Phe Thr Gly Val GlnAsn Phe Ser Lys 215 220 225 230 atc cac ctg gtg gac aga tgg aat ggg ctcagc aag gtc aac tac tgg 893 Ile His Leu Val Asp Arg Trp Asn Gly Leu SerLys Val Asn Tyr Trp 235 240 245 cat tca gag cag tgc aac atg atc aat ggcact tcc ggg cag atg tgg 941 His Ser Glu Gln Cys Asn Met Ile Asn Gly ThrSer Gly Gln Met Trp 250 255 260 gca cca ttc atg aca ccc cag tcc tcg ctggaa ttc ttc agt ccg gaa 989 Ala Pro Phe Met Thr Pro Gln Ser Ser Leu GluPhe Phe Ser Pro Glu 265 270 275 gcc tgc agg tct atg aag ctc acc tac catgat tca ggg gtg ttt gaa 1037 Ala Cys Arg Ser Met Lys Leu Thr Tyr His AspSer Gly Val Phe Glu 280 285 290 ggc atc ccc acc tat cgc ttc aca gcc cctaaa act ttg ttt gcc aat 1085 Gly Ile Pro Thr Tyr Arg Phe Thr Ala Pro LysThr Leu Phe Ala Asn 295 300 305 310 ggg tct gtt tac cca ccc aat gaa ggtttc tgc ccg tgc ctt gaa tcc 1133 Gly Ser Val Tyr Pro Pro Asn Glu Gly PheCys Pro Cys Leu Glu Ser 315 320 325 ggc att caa aat gtc agc act tgc aggttt ggt gca ccc ctg ttt ctg 1181 Gly Ile Gln Asn Val Ser Thr Cys Arg PheGly Ala Pro Leu Phe Leu 330 335 340 tca cac cct cac ttc tac aat gca gaccct gtg cta tca gaa gcc gtt 1229 Ser His Pro His Phe Tyr Asn Ala Asp ProVal Leu Ser Glu Ala Val 345 350 355 ctg ggt ctg aac cct gac cca agg gagcat tct ttg ttc ctt gac atc 1277 Leu Gly Leu Asn Pro Asp Pro Arg Glu HisSer Leu Phe Leu Asp Ile 360 365 370 cat ccg gtc act ggg atc ccc atg aactgt tct gtg aag ttg cag ata 1325 His Pro Val Thr Gly Ile Pro Met Asn CysSer Val Lys Leu Gln Ile 375 380 385 390 agc ctc tac atc aaa gct gtc aagggc att ggg caa aca ggg aag atc 1373 Ser Leu Tyr Ile Lys Ala Val Lys GlyIle Gly Gln Thr Gly Lys Ile 395 400 405 gag ccc gtg gtc ctc cca ttg ctgtgg ttt gag cag agc ggt gcc atg 1421 Glu Pro Val Val Leu Pro Leu Leu TrpPhe Glu Gln Ser Gly Ala Met 410 415 420 ggc ggc gag ccc ctg aac acg ttctac acg cag ctg gtg ctg atg ccc 1469 Gly Gly Glu Pro Leu Asn Thr Phe TyrThr Gln Leu Val Leu Met Pro 425 430 435 cag gta ctt cag tat gtg cag tatgtg ctg ctg ggg ctg ggc ggc ctc 1517 Gln Val Leu Gln Tyr Val Gln Tyr ValLeu Leu Gly Leu Gly Gly Leu 440 445 450 ctg ctg ctg gtg ccc gtc atc taccag ttg cgc agc cag gag aaa tgc 1565 Leu Leu Leu Val Pro Val Ile Tyr GlnLeu Arg Ser Gln Glu Lys Cys 455 460 465 470 ttt tta ttt tgg agt ggt agtaaa aag ggc tcg cag gat aag gag gcc 1613 Phe Leu Phe Trp Ser Gly Ser LysLys Gly Ser Gln Asp Lys Glu Ala 475 480 485 att cag gcc tac tct gag tctctg atg tca cca gct gcc aag ggc acg 1661 Ile Gln Ala Tyr Ser Glu Ser LeuMet Ser Pro Ala Ala Lys Gly Thr 490 495 500 gtg ctg caa gaa gcc aag ctgtagggtccca aagacaccac gagccccccc 1712 Val Leu Gln Glu Ala Lys Leu 505aacctgatag cttggtcaga ccagccatcc agcccctaca ccccgcttct tgaggactct 1772ctcagcggac agtccgccag tgccatggcc tgagccccag atgtcacacc tgt 1825 4 509PRT Human 4 Met Gly Gly Ser Ala Arg Ala Arg Trp Val Ala Val Gly Leu GlyVal 1 5 10 15 Val Gly Leu Leu Cys Ala Val Leu Gly Val Val Met Ile LeuVal Met 20 25 30 Pro Ser Leu Ile Lys Gln Gln Val Leu Lys Asn Val Arg IleAsp Pro 35 40 45 Ser Ser Leu Ser Phe Ala Met Trp Lys Glu Ile Pro Val ProPhe Tyr 50 55 60 Leu Ser Val Tyr Phe Phe Glu Val Val Asn Pro Ser Glu IleLeu Lys 65 70 75 80 Gly Glu Lys Pro Val Val Arg Glu Arg Gly Pro Tyr ValTyr Arg Glu 85 90 95 Phe Arg His Lys Ala Asn Ile Thr Phe Asn Asp Asn AspThr Val Ser 100 105 110 Phe Val Glu His Arg Ser Leu His Phe Gln Pro AspArg Ser His Gly 115 120 125 Ser Glu Ser Asp Tyr Ile Ile Leu Pro Asn IleLeu Val Leu Gly Gly 130 135 140 Ala Val Met Met Glu Ser Lys Ser Ala GlyLeu Lys Leu Met Met Thr 145 150 155 160 Leu Gly Leu Ala Thr Leu Gly GlnArg Ala Phe Met Asn Arg Thr Val 165 170 175 Gly Glu Ile Leu Trp Gly TyrGlu Asp Pro Phe Val Asn Phe Ile Asn 180 185 190 Lys Tyr Leu Pro Asp MetPhe Pro Ile Lys Gly Lys Phe Gly Leu Phe 195 200 205 Val Glu Met Asn AsnSer Asp Ser Gly Leu Phe Thr Val Phe Thr Gly 210 215 220 Val Gln Asn PheSer Lys Ile His Leu Val Asp Arg Trp Asn Gly Leu 225 230 235 240 Ser LysVal Asn Tyr Trp His Ser Glu Gln Cys Asn Met Ile Asn Gly 245 250 255 ThrSer Gly Gln Met Trp Ala Pro Phe Met Thr Pro Gln Ser Ser Leu 260 265 270Glu Phe Phe Ser Pro Glu Ala Cys Arg Ser Met Lys Leu Thr Tyr His 275 280285 Asp Ser Gly Val Phe Glu Gly Ile Pro Thr Tyr Arg Phe Thr Ala Pro 290295 300 Lys Thr Leu Phe Ala Asn Gly Ser Val Tyr Pro Pro Asn Glu Gly Phe305 310 315 320 Cys Pro Cys Leu Glu Ser Gly Ile Gln Asn Val Ser Thr CysArg Phe 325 330 335 Gly Ala Pro Leu Phe Leu Ser His Pro His Phe Tyr AsnAla Asp Pro 340 345 350 Val Leu Ser Glu Ala Val Leu Gly Leu Asn Pro AspPro Arg Glu His 355 360 365 Ser Leu Phe Leu Asp Ile His Pro Val Thr GlyIle Pro Met Asn Cys 370 375 380 Ser Val Lys Leu Gln Ile Ser Leu Tyr IleLys Ala Val Lys Gly Ile 385 390 395 400 Gly Gln Thr Gly Lys Ile Glu ProVal Val Leu Pro Leu Leu Trp Phe 405 410 415 Glu Gln Ser Gly Ala Met GlyGly Glu Pro Leu Asn Thr Phe Tyr Thr 420 425 430 Gln Leu Val Leu Met ProGln Val Leu Gln Tyr Val Gln Tyr Val Leu 435 440 445 Leu Gly Leu Gly GlyLeu Leu Leu Leu Val Pro Val Ile Tyr Gln Leu 450 455 460 Arg Ser Gln GluLys Cys Phe Leu Phe Trp Ser Gly Ser Lys Lys Gly 465 470 475 480 Ser GlnAsp Lys Glu Ala Ile Gln Ala Tyr Ser Glu Ser Leu Met Ser 485 490 495 ProAla Ala Lys Gly Thr Val Leu Gln Glu Ala Lys Leu 500 505 5 1002 DNA Human5 actgcggaga tgagggtcta gaaggtggtg gcggggcatg tggaccgttg taagggctct 60ggggttcctg ggtgggctgg cgaagtccta ctcacagtga ccaaccatga tgatggtccc 120gatagaggag gagagggagg aggagggaaa aggaagggtg aggggctcag aggggagagc 180tgggaggagg ggagacatag gtgggggaag gggtaggaga aaggggaagg gagcaagagg 240gtgaggggca ccaggcccca tagacgtttt ggctcagcgg ccacgaggct tcatcagctc 300ccgccccaaa acggaagcga ggccgtgggg gcagcggcag catggcgggg cttgtcttgg 360cggccatggc cccgccccct gcccgtccga tcagcgcccc gccccgtccc cgccccgacc 420ccgccccggg cccgctcagg ccccgcccct gccgccggaa tcctgaagcc caaggctgcc 480cgggggcggt ccggcggcgc cggcgatggg gcataaaacc actggccacc tgccgggctg 540ctcctgcgtg cgctgccgtc ccggatccac cgtgcctctg cggcctgcgt gccccgagtc 600cccgcctgtg tcgtctctgt cgccgtcccc gtctcctgcc aggcgcggag ccctgcgagc 660cgcgggtggg ccccaggcgc gcagacatgg gctgctccgc caaagcgcgc tgggctgccg 720gggcgctggg cgtcgcgggg ctactgtgcg ctgtgctggg cgctgtcatg atcgtgatgg 780tgccgtcgct catcaagcag caggtcctta aggtgggtga gggagacccc agggggtccg 840cgcacggacc cgggctgttg ggcgctgggc gccgggagga cccgcgcgtt gcggtgggtg 900ggcgaccgca gcggaatcgg cgcccgggcc tggcgccgca gaacacgagg gaggccaggc 960gcttcgggag gggctgctgc ccgcctcccc accaccctca cc 1002 6 479 DNA Human 6agcctcatgt gcgaagggct ttcccaccac ctcctatccc aagctcccgc cgaggagccc 60cttccctggc cgggctcggg cagctgttcc ggagccttgt ggtggggcgt ggggccctca 120tcactctcct cacaagcgta cttgtccctt cccctgcaga acgtgcgcat cgaccccagt 180agcctgtcct tcaacatgtg gaaggagatc cctatcccct tctatctctc cgtctacttc 240tttgacgtca tgaaccccag cgagatcctg aagggcgaga agccgcaggt gcgggagcgc 300gggccctacg tgtacaggtg aggctgtgtc cacgtgatgg tggacgggcc ggctgacgct 360gggcatggga cgggtctcaa gtggacggga tggggaggct gctgactgac ccccaaacat 420tgttccggaa gcacgcaact catagtcggg gtaagtgcta ctcccaaaaa agtttgcgt 479 7495 DNA Human 7 catgtcctgc agtgggcagg cagcgggagg gacagacttg gcgaaggggccgagctcagc 60 tttggctgtg gggccggagg tgtgcacaga cgtccagggc ccctggttcccaggcaggca 120 ttgcaggcga gtagaaggga aacgtcccat gcagcggggc ggggcgtctgacccactggc 180 ttcccccaca gggagttcag gcacaaaagc aacatcacct tcaacaacaacgacaccgtg 240 tccttcctcg agtaccgcac cttccagttc cagccctcca agtcccacggctcggagagc 300 gactacatcg tcatgcccaa catcctggtc ttggtgaggc tgccctgtggcccacgccgc 360 ctcgcaccct gacctcgtcc cctgtctctc ctcccgcctg ccccttgtgcagagagcagt 420 ccctgaggtg gtcggagcgt ggggactcac gcctggtggg tggctttcggccctgtgctg 480 tctccaccac cccca 495 8 526 DNA Human 8 ggtggttctggtgtcccaga tgccccacgt ggccactcca ggggcctcct gcaccccagc 60 atttcccttcatgggctctt tgctgtgagg cccagctggg gccaagggag gatgggccag 120 ccacgtccagcctctgacac tagtgtccct tcgccttgca gggtgcggcg gtgatgatgg 180 agaataagcccatgaccctg aagctcatca tgaccttggc attcaccacc ctcggcgaac 240 gtgccttcatgaaccgcact gtgggtgaga tcatgtgggg ctacaaggac cccttgtgaa 300 tctcatcaacaagtactttc caggcatgtt ccccttcaag gacaagttcg gattatttgc 360 tgaggtacgtgtggcctggt gagaagccaa agattcaggc ctgtgtcctg tcttcccctc 420 acacagcctggacactggtc accagcttgc tttgtagctg gctggggatc tagtggctgt 480 gggttgtaagtgactgagaa cctgactcaa accggcttga gtgaaa 526 9 416 DNA Human 9 cctctcggtccccagacact gggcatttgg cagtgaacca gatgctgggg gccctgtcct 60 tctggtggagggggaggagg gctcagccca gaatgttcag accaggccgg ctcaatggca 120 ggcctaagccttacgatgct gttccctgct gtgtctgtag ctcaacaact ccgactctgg 180 gctcttcacggtgttcacgg gggtccagaa catcagcagg atccacctcg tggacaagtg 240 gaacgggctgagcaaggtga ggggcgagag gcgagggccc ctgtcgccag ggagagggga 300 gggtgggcccggccatggct gctcgggagt ggcagggacc agagagctcc ttcttccttt 360 gtcgtgaagagggtgctggg aggatgaaca ctcttgaagt tggaggaggg atttta 416 10 436 DNA Human10 tctctgtgtg tctacatagc ctgccctctt cccaccgtgc cagtattggg aattgagtgg 60ccgtgcgtgc accagggtga gttaggtgtg cagcacctga gagggcttat taaggggcct 120tggccctact gaggggtcta gtctggatgc ttccccccag gttgacttct ggcattccga 180tcagtgcaac atgatcaatg gaacttctgg gcaaatgtgg ccgcccttca tgactcctga 240gtcctcgctg gagttctaca gcccggaggc ctgccggtaa tcactgggac tcggggcctc 300ctgggtttcc tgggtagctc atggccaaat tctgtggtgt tggctgtgca cttggaaagc 360attttgactc atcgtggatt tgactcagta gcccttggca ccagcttgaa ttctctttgg 420tcacaccacc aaaagc 436 11 481 DNA Human All occurrences of n =anynucleotide 11 ggaggtcgct gcagctccgc gggtgagaga tgggggcggt ttggacccgggaggtggtag 60 cgcccgtggg gagaagtggc tggatctggg cagcctttgg cagggcctggctctggccgc 120 cgggtctggg tgtcccctct catcctgtct gtcccctgca gatccatgaagctaatgtac 180 aaggagtcag gggtgtttga aggcatcccc acctatcgct tcgtggctcccaaaaccctg 240 tttgccaacg ggtccatcta cccacccaac gaaggcttct gcccgtgcctggagtctgga 300 attcagaacg tcagcagctg caggttcagt acgtgccgtc ccctgttctgggatngccgg 360 agggtgttag gtntngggca cctnanggtt tatctgccca atgctgtctgcttaatctct 420 ggcctctgta ctcttgataa cccattaagc caaaaatatg atgcctctgggacgatatct 480 g 481 12 430 DNA Human 12 tggggctttt tacagaatggaggaagggat cctctctgtc gggtattatg gtcatcgcca 60 cgggggtgcc gtgcagaccacagctctgtg cagacttccg gagtggcagg acgtgccaat 120 atactgtcgt tgtatgatgtcccctccctg cccttgttgt aggtgccccc ttgtttctct 180 cccatcctca cttcatcaacgccgacccgg ttctggcaga agcggtgact ggcctgcacc 240 ctaaccagga ggcacactccttgttcgtgg acatccaccc ggtgagcccc tgccatcctc 300 tgtggggggt gggtgattcctggttggagc acacctggct gcctcctctc tccccaggca 360 gagagctgct gtgggctggggtggtgggaa gcctggcttc tagaatctcg agccaccaaa 420 gttccttact 430 13 390DNA Human All occurrences of n =any nucleotide 13 ccccagcctg tggcttgttttaggtaagat acaagcaagc tccactgggc agttagctgg 60 gacgcccacc ctcttgactgggaccaggga aaagaaggtt gactgtgtcc ctggagcttg 120 ggggtggcca gtctcctcactgtgtttgtt gccgcaggtc acgggaatcc ccatgaactg 180 ctctgtgaaa ctgcagctgagcctctacat gaaatctgtc gcaggcattg ggtgagtggg 240 gactgggaac tggggctgcattgctcattg agagattang tgctcagtgc tccagtgttc 300 ccagactccc ctgacataccccaggaaaca gggcatgggg aagggagagg gtcctattgg 360 gggtggaatc cagtccctgctgatcttctc 390 14 370 DNA Human 14 atggctccta aagtgtttca gctcattgtttatatttggt ggtgagggtt tagtgtgtgc 60 aaaattatac taaacctgtt tagatgttgtattcaagcag aattagatca agtttgggtg 120 taagactttg ttccaacacc tatgtcttgcttatttccag acaaactggg aagattgagc 180 ctgtggtcct gccgctgctc tggtttgcagaggtaagggt gcgttgggca cagcgtcggg 240 ggcttttgtt aatagccaat gtgggcatttgaggcaggag gcggggggag caccttgtag 300 aaagggagag ggctgagcca gggtaaccggactgttacat ggaccagcgt atcatacact 360 tcaccctgtc 370 15 470 DNA Human 15cctggaggga ggaggtccct ggcaggctcc aacacatgct ttagccggga agcttgaggt 60ggggaaaagc tgaggcgggc acagaggaag gtgttgggtg gcatctgcgc tgtagcccgc 120agcctgcggc cccagctcat gtgtttgtca ttctgtctcc tcagagcggg gccatggagg 180gggagactct tcacacattc tacactcagc tggtgttgat gcccaaggtg atgcactatg 240cccagtacgt cctcctggcg ctgggctgcg tcctgctgct ggtccctgtc atctgccaaa 300tccggagcca agtaggtgct ggccagaggg cagcccgggc tgacagccat tcgcttgcct 360gctgggggaa aggggcctca gatcggaccc tctggccaac cgcagcctgg agcccacctc 420cagcagcagt cctgcgtctc tgccggagtg ggagcggtca ctgctggggg 470 16 450 DNAHuman 16 ccccacatct cagccacctg caatcgttga gggttgttgg actctaaacttatgtgcctt 60 tcctgtttcc tctttgcctt ttgcaaattg aagaaccgtg taaaaccatttttatgtggc 120 ttcaacgtca actataaatt agcttggtta tcttctagga gaaatgctatttattttgga 180 gtagtagtaa aaagggctca aaggataagg aggccattca ggcctattctgaatccctga 240 tgacatcagc tcccaagggc tctgtgctgc aggaagcaaa actgtaggtgggtaccaggt 300 aatgccgtgc gcctccccgc cccctcccat atcaagtaga atgctggcggcttaaaacat 360 ttggggtcct gctcattcct tcagcctcaa cttcacctgg agtgtctacagactgaagat 420 gcatatttgt gtattttgct tttggagaaa 450 17 544 DNA Human 17actgcggaga tgagggtcta gaaggtggtg gcggggcatg tggaccgttg taagggctct 60ggggttcctg ggtgggctgg cgaagtccta ctcacagtga ccaaccatga tgatggtccc 120gatagaggag gagagggagg aggagggaaa aggaagggtg aggggctcag aggggagagc 180tgggaggagg ggagacatag gtgggggaag gggtaggaga aaggggaagg gagcaagagg 240gtgaggggca ccaggcccca tagacgtttt ggctcagcgg ccacgaggct tcatcagctc 300ccgccccaaa acggaagcga ggccgtgggg gcagcggcag catggcgggg cttgtcttgg 360cggccatggc cccgccccct gcccgtccga tcagcgcccc gccccgtccc cgccccgacc 420ccgccccggg cccgctcagg ccccgcccct gccgccggaa tcctgaagcc caaggctgcc 480cgggggcggt ccggcggcgc cggcgatggg gcataaaacc actggccacc tgccgggctg 540ctcc 544 18 190 DNA Human 18 gtgggtgagg gagaccccag ggggtccgcg cacggacccgggctgttggg cgctgggcgc 60 cgggaggacc cgcgcgttgc ggtgggtggg cgaccgcagcggaatcggcg cccgggcctg 120 gcgccgcaga acacgaggga ggccaggcgc ttcgggaggggctgctgccc gcctccccac 180 caccctcacc 190 19 159 DNA Human 19 agcctcatgtgcgaagggct ttcccaccac ctcctatccc aagctcccgc cgaggagccc 60 cttccctggccgggctcggg cagctgttcc ggagccttgt ggtggggcgt ggggccctca 120 tcactctcctcacaagcgta cttgtccctt cccctgcag 159 20 162 DNA Human 20 gtgaggctgtgtccacgtga tggtggacgg gccggctgac gctgggcatg ggacgggtct 60 caagtggacgggatggggag gctgctgact gacccccaaa cattgttccg gaagcacgca 120 actcatagtcggggtaagtg ctactcccaa aaaagtttgc gt 162 21 191 DNA Human 21 catgtcctgcagtgggcagg cagcgggagg gacagacttg gcgaaggggc cgagctcagc 60 tttggctgtggggccggagg tgtgcacaga cgtccagggc ccctggttcc caggcaggca 120 ttgcaggcgagtagaaggga aacgtcccat gcagcggggc ggggcgtctg acccactggc 180 ttcccccaca g191 22 162 DNA Human 22 gtgaggctgc cctgtggccc acgccgcctc gcaccctgacctcgtcccct gtctctcctc 60 ccgcctgccc cttgtgcaga gagcagtccc tgaggtggtcggagcgtggg gactcacgcc 120 tggtgggtgg ctttcggccc tgtgctgtct ccaccacccc ca162 23 161 DNA Human 23 ggtggttctg gtgtcccaga tgccccacgt ggccactccaggggcctcct gcaccccagc 60 atttcccttc atgggctctt tgctgtgagg cccagctggggccaagggag gatgggccag 120 ccacgtccag cctctgacac tagtgtccct tcgccttgca g161 24 162 DNA Human 24 gtacgtgtgg cctggtgaga agccaaagat tcaggcctgtgtcctgtctt cccctcacac 60 agcctggaca ctggtcacca gcttgctttg tagctggctggggatctagt ggctgtgggt 120 tgtaagtgac tgagaacctg actcaaaccg gcttgagtga aa162 25 160 DNA Human 25 cctctcggtc cccagacact gggcatttgg cagtgaaccagatgctgggg gccctgtcct 60 tctggtggag ggggaggagg gctcagccca gaatgttcagaccaggccgg ctcaatggca 120 ggcctaagcc ttacgatgct gttccctgct gtgtctgtag160 26 160 DNA Human 26 gtgaggggcg agaggcgagg gcccctgtcg ccagggagaggggagggtgg gcccggccat 60 ggctgctcgg gagtggcagg gaccagagag ctccttcttcctttgtcgtg aagagggtgc 120 tgggaggatg aacactcttg aagttggagg agggatttta160 27 160 DNA Human 27 tctctgtgtg tctacatagc ctgccctctt cccaccgtgccagtattggg aattgagtgg 60 ccgtgcgtgc accagggtga gttaggtgtg cagcacctgagagggcttat taaggggcct 120 tggccctact gaggggtcta gtctggatgc ttccccccag160 28 160 DNA Human 28 gtaatcactg ggactcgggg cctcctgggt ttcctgggtagctcatggcc aaattctgtg 60 gtgttggctg tgcacttgga aagcattttg actcatcgtggatttgactc agtagccctt 120 ggcaccagct tgaattctct ttggtcacac caccaaaagc160 29 161 DNA Human 29 ggaggtcgct gcagctccgc gggtgagaga tgggggcggtttggacccgg gaggtggtag 60 cgcccgtggg gagaagtggc tggatctggg cagcctttggcagggcctgg ctctggccgc 120 cgggtctggg tgtcccctct catcctgtct gtcccctgca g161 30 153 DNA Human All occurrences of n =any nucleotide 30 gtacgtgccgtcccctgttc tgggatngcc ggagggtgtt aggtntnggg cacctnangg 60 tttatctgcccaatgctgtc tgcttaatct ctggcctctg tactcttgat aacccattaa 120 gccaaaaatatgatgcctct gggacgatat ctg 153 31 162 DNA Human 31 tggggctttt tacagaatggaggaagggat cctctctgtc gggtattatg gtcatcgcca 60 cgggggtgcc gtgcagaccacagctctgtg cagacttccg gagtggcagg acgtgccaat 120 atactgtcgt tgtatgatgtcccctccctg cccttgttgt ag 162 32 149 DNA Human 32 gtgagcccct gccatcctctgtggggggtg ggtgattcct ggttggagca cacctggctg 60 cctcctctct ccccaggcagagagctgctg tgggctgggg tggtgggaag cctggcttct 120 agaatctcga gccaccaaagttccttact 149 33 157 DNA Human 33 ccccagcctg tggcttgttt taggtaagatacaagcaagc tccactgggc agttagctgg 60 gacgcccacc ctcttgactg ggaccagggaaaagaaggtt gactgtgtcc ctggagcttg 120 ggggtggcca gtctcctcac tgtgtttgttgccgcag 157 34 159 DNA Human All occurrences of n =any nucleotide 34gtgagtgggg actgggaact ggggctgcat tgctcattga gagattangt gctcagtgct 60ccagtgttcc cagactcccc tgacataccc caggaaacag ggcatgggga agggagaggg 120tcctattggg ggtggaatcc agtccctgct gatcttctc 159 35 160 DNA Human 35atggctccta aagtgtttca gctcattgtt tatatttggt ggtgagggtt tagtgtgtgc 60aaaattatac taaacctgtt tagatgttgt attcaagcag aattagatca agtttgggtg 120taagactttg ttccaacacc tatgtcttgc ttatttccag 160 36 158 DNA Human 36gtaagggtgc gttgggcaca gcgtcggggg cttttgttaa tagccaatgt gggcatttga 60ggcaggaggc ggggggagca ccttgtagaa agggagaggg ctgagccagg gtaaccggac 120tgttacatgg accagcgtat catacacttc accctgtc 158 37 164 DNA Human 37cctggaggga ggaggtccct ggcaggctcc aacacatgct ttagccggga agcttgaggt 60ggggaaaagc tgaggcgggc acagaggaag gtgttgggtg gcatctgcgc tgtagcccgc 120agcctgcggc cccagctcat gtgtttgtca ttctgtctcc tcag 164 38 159 DNA Human 38gtaggtgctg gccagagggc agcccgggct gacagccatt cgcttgcctg ctgggggaaa 60ggggcctcag atcggaccct ctggccaacc gcagcctgga gcccacctcc agcagcagtc 120ctgcgtctct gccggagtgg gagcggtcac tgctggggg 159 39 158 DNA Human 39ccccacatct cagccacctg caatcgttga gggttgttgg actctaaact tatgtgcctt 60tcctgtttcc tctttgcctt ttgcaaattg aagaaccgtg taaaaccatt tttatgtggc 120ttcaacgtca actataaatt agcttggtta tcttctag 158 40 163 DNA Human 40gtgggtacca ggtaatgccg tgcgcctccc cgccccctcc catatcaagt agaatgctgg 60cggcttaaaa catttggggt cctgctcatt ccttcagcct caacttcacc tggagtgtct 120acagactgaa gatgcatatt tgtgtatttt gcttttggag aaa 163 41 23 DNA Human 41cccctgccgc cggaatcctg aag 23 42 24 DNA Human 42 cgctttggcg gagcagcccatgtc 24 43 24 DNA Human 43 tggggccctc atcactctcc tcac 24 44 23 DNA Human44 gcagcctccc catcccgtcc act 23 45 18 DNA Human 45 attgcaggcg agtagaag18 46 18 DNA Human 46 caggcgggag gagagaca 18 47 20 DNA Human 47tgggctcttt gctgtgaggc 20 48 20 DNA Human 48 ccaggctgtg tgaggggaag 20 4920 DNA Human 49 gcccagaatg ttcagaccag 20 50 20 DNA Human 50 gcaccctcttcacgacaaag 20 51 19 DNA Human 51 cacctgagag ggcttatta 19 52 19 DNA Human52 caaaatgctt tccaagtgc 19 53 20 DNA Human 53 gccgccgggt ctgggtgtcc 2054 23 DNA Human 54 cagaggccag agattaagca gac 23 55 20 DNA Human 55ttgtatgatg tcccctccct 20 56 20 DNA Human 56 ttcccaccac cccagcccac 20 5720 DNA Human 57 ggttgactgt gtccctggag 20 58 21 DNA Human 58 gggaacactggagcactgag c 21 59 20 DNA Human 59 ggtggtgagg gtttagtgtg 20 60 20 DNAHuman 60 ctccccccgc ctcctgcctc 20 61 20 DNA Human 61 aaggtgttgggtggcatctg 20 62 20 DNA Human 62 ggctccaggc tgcggttggc 20 63 19 DNAHuman 63 ttgaagaacc gtgtaaaac 19 64 18 DNA Human 64 ttgaggctga aggaatga18 65 430 DNA Human 65 tggggctttt tacagaatgg aggaagggat cctctctgtcgggtattatg gtcatcgcca 60 cgggggtgcc gtgcagacca cagctctgtg cagacttccggagtggcagg acgtgccaat 120 atactgtcgt tgtatgatgt cccctccctg cccttgttgtaggtgccccc ttgtttctct 180 cccatcctca cttcatcaac gctgacccgg ttctggcagaagcggtgact ggcctgcacc 240 ctaaccagga ggcacactcc ttgttcgtgg acatccacccggtgagcccc tgccatcctc 300 tgtggggggt gggtgattcc tggttggagc acacctggctgcctcctctc tccccaggca 360 gagagctgct gtgggctggg gtggtgggaa gcctggcttctagaatctcg agccaccaaa 420 gttccttact 430 66 160 DNA Human 66 gtgaggggcgagaggcgagg gcccctgtcg ccagggagag gggagggtgg gcctggccat 60 ggctgctcgggagtggcagg gaccagagag ctccttcttc ctttgtcgtg aagagggtgc 120 tgggaggatgaacactcttg aagttggagg agggatttta 160 67 20 DNA Human 67 aaccgggtcagcgttgagga 20 68 31 DNA Human 68 tgccagaacc gggtcagcgt tgaggaagtg a 3169 20 DNA Human 69 tcctcaacgc tgacccggtt 20 70 31 DNA Human 70tcacttcctc aacgctgacc cggttctggc a 31 71 20 DNA Human 71 aaccgggtcggcgttgatga 20 72 31 DNA Human 72 tgccagaacc gggtcggcgt tgatgaagtg a 3173 20 DNA Human 73 tcatcaacgc cgacccggtt 20 74 31 DNA Human 74tcacttcatc aacgccgacc cggttctggc a 31 75 21 DNA Human 75 agccatggccgggcccaccc t 21 76 31 DNA Human 76 cgagcagcca tggccgggcc caccctcccc t 3177 21 DNA Human 77 agggtgggcc cggccatggc t 21 78 31 DNA Human 78aggggagggt gggcccggcc atggctgctc g 31 79 21 DNA Human 79 agccatggccaggcccaccc t 21 80 31 DNA Human 80 cgagcagcca tggccaggcc caccctcccc t 3181 21 DNA Human 81 agggtgggcc tggccatggc t 21 82 31 DNA Human 82aggggagggt gggcctggcc atggctgctc g 31 83 22 DNA Human 83 tcctgggtgggctggcgaag tc 22 84 24 DNA Human 84 gttttggggc gggagctgat gaag 24 85 18DNA Human 85 tgtaaaacga cggccagt 18 86 18 DNA Human 86 caggaaacagctatgacc 18 87 62 DNA Human 87 ctgagcaagg tgaggggcga gaggcgagggcccctgtcgc cagggagggg agggtgggcc 60 yg 62 88 51 DNA Human 88 cstgcggccccagctcatgt gtttgtcatt ctgtctcctc agagcggggc c 51 89 24 DNA Human 89ccggcgatgg ggcataaaac cact 24 90 23 DNA Human 90 cgcccagcac agcgcacagtagc 23 91 20 DNA Human 91 gcccagaatg ttcagaccag 20 92 20 DNA Human 92gcaccctctt cacgacaaag 20 93 34 DNA Human 93 ccttgtttct ctcccatcctcacttcctca aggc 34 94 22 DNA Human 94 caccacccca gcccacagca gc 22 951002 DNA Human 95 actgcggaga tgagggtcta gaaggtggtg gcggggcatg tggaccgttgtaagggctct 60 ggggttcctg ggtgggctgg cgaagtccta ctcacagtga ccaaccatgatgatggtccc 120 gatagaggag gagagggagg aggagggaaa aggaagggtg aggggctcagaggggagagc 180 tgggaggagg ggagacatag gtgggggaag gggtaggaga aaggggaagggagcaagagg 240 gtgaggggca ccaggcccca tagacgtttt ggctcagcgg ccacgaggcttcatcagctc 300 ccgccccaaa acggaagcga ggccgtgggg gcagcggcag catggcggggcttgtcttgg 360 cggccatggc cccgccccct gcccgtccga tcagcgcccc gccccgtccccgccccgacc 420 ccgccccggg cccgctcagg ccccgcccct gccgccggaa tcctgaagcccaaggctgcc 480 cgggggcggt ccggcggcgc cggcgatggg gcataaaacc actggccacctgccgggctg 540 ctcctgcgtg cgctgccgtc ccggatccac cgtgcctctg cggcctgcgtgccccgagtc 600 cccgcctgtg tcgtctctgt cgccgtcccc gtctcctgcc aggcgcggagccctgcgagc 660 cgcgggtggg ccccaggcgc gcagacatga gctgctccgc caaagcgcgctgggctgccg 720 gggcgctggg cgtcgcgggg ctactgtgcg ctgtgctggg cgctgtcatgatcgtgatgg 780 tgccgtcgct catcaagcag caggtcctta aggtgggtga gggagaccccagggggtccg 840 cgcacggacc cgggctgttg ggcgctgggc gccgggagga cccgcgcgttgcggtgggtg 900 ggcgaccgca gcggaatcgg cgcccgggcc tggcgccgca gaacacgagggaggccaggc 960 gcttcgggag gggctgctgc ccgcctcccc accaccctca cc 1002 96495 DNA Human 96 catgtcctgc agtgggcagg cagcgggagg gacagacttg gcgaaggggccgagctcagc 60 tttggctgtg gggccggagg tgtgcacaga cgtccagggc ccctggttcccaggcaggca 120 ttgcaggcga gtagaaggga aacgtcccat gcagcggggc ggggcgtctgacccactggc 180 ttcccccaca gggagttcag gcacaaaagc aacatcacct tcaacaacaacgacaccgtg 240 tccttcctcg agtaccgcac cttccagttc cagccctcca agtcccacggctcggagagc 300 gactacatca tcatgcccaa catcctggtc ttggtgaggc tgccctgtggcccacgccgc 360 ctcgcaccct gacctcgtcc cctgtctctc ctcccgcctg ccccttgtgcagagagcagt 420 ccctgaggtg gtcggagcgt ggggactcac gcctggtggg tggctttcggccctgtgctg 480 tctccaccac cccca 495 97 470 DNA Human 97 cctggagggaggaggtccct ggcaggctcc aacacatgct ttagccggga agcttgaggt 60 ggggaaaagctgaggcgggc acagaggaag gtgttgggtg gcatctgcgc tgtagcccgc 120 agcgtgcggccccagctcat gtgtttgtca ttctgtctcc tcagagcggg gccatggagg 180 gggagactcttcacacattc tacactcagc tggtgttgat gcccaaggtg atgcactatg 240 cccagtacgtcctcctggcg ctgggctgcg tcctgctgct ggtccctgtc atctgccaaa 300 tccggagccaagtaggtgct ggccagaggg cagcccgggc tgacagccat tcgcttgcct 360 gctgggggaaaggggcctca gatcggaccc tctggccaac cgcagcctgg agcccacctc 420 cagcagcagtcctgcgtctc tgccggagtg ggagcggtca ctgctggggg 470 98 21 DNA Human 98gcggagcagc tcatgtctgc g 21 99 31 DNA Human 99 ctttcgcgga gcagctcatgtctgcgcgcc t 31 100 21 DNA Human 100 cgcagacatg agctgctccg c 21 101 31DNA Human 101 aggcgcgcag acatgagctg ctccgccaaa g 31 102 21 DNA Human 102gcggagcagc gcatgtctgc g 21 103 31 DNA Human 103 ctttcgcgga gcagcgcatgtctgcgcgcc t 31 104 21 DNA Human 104 cgcagacatg cgctgctccg c 21 105 31DNA Human 105 aggcgcgcag acatgcgctg ctccgccaaa g 31 106 21 DNA Human 106ttgggcatga tgatgtagac g 21 107 31 DNA Human 107 ggatgttggg catgatgatgtagacgctct c 31 108 21 DNA Human 108 cgactacatc atcatgccca a 21 109 31DNA Human 109 gagagcgact acatcatcat gcccaacatc c 31 110 21 DNA Human 110ttgggcatga ggatgtagac g 21 111 31 DNA Human 111 ggatgttggg catgaggatgtagacgctct c 31 112 21 DNA Human 112 cgactacatc ctcatgccca a 21 113 32DNA Human 113 gagagcgact acatccatca tgcccaacat cc 32 114 21 DNA Human114 tggggccgca cgctgcgggc t 21 115 31 DNA Human 115 tgagctggggccgcacgctg cgggctacag c 31 116 21 DNA Human 116 agcccgcagc gtgcggcccc a21 117 31 DNA Human 117 gctgtagccc gcagcgtgcg gccccagctc a 31 118 21 DNAHuman 118 tggggccgca ggctgcgggc t 21 119 31 DNA Human 119 tgagctggggccgcaggctg cgggctacag c 31 120 21 DNA Human 120 agcccgcagc ctgcggcccc a21 121 31 DNA Human 121 gctgtagccc gcagcctgcg gccccagctc a 31

1. A method for determining whether a subject has, or is at risk ofdeveloping, an abnormally low HDL level, comprising determining theidentity of the allelic variant of a polymorphic region of the SR-BIgene of the subject and comparing the allelic variant of the subjectwith allelic variants associated with abnormally low HDL levels, tothereby determine whether the subject has an allelic variant of apolymorphic region of an SR-BI gene associated with a abnormally low HDLlevel.
 2. A method of claim 1, wherein the polymorphic region is locatedin an exon.
 3. A method of claim 2, wherein the intron is exon
 8. 4. Amethod of claim 3, wherein the polymorphic region is a nucleotidepolymorphism.
 5. A method of claim 4, wherein the nucleotidepolymorphism is located at position 41 of exon
 8. 6. A method of claim5, wherein nucleotide 41 of exon 8 of the SR-BI gene in a normal subjectis a thymidine and the presence of a nucleotide other than a thymidineat position 41 of exon 8 in the SR-BI gene of a subject indicates thatthe subject has or is at risk of developing an abnormally low HDL level.7. A method of claim 6, wherein the nucleotide other than a thymidine atposition 41 of exon 8 is a cytidine.
 8. A method of claim 1, whereindetermining the identity of the allelic variant of a polymorphic regionof an SR-BI gene comprises determining the identity of at least onenucleotide of the polymorphic region.
 9. A method of claim 2, whereindetermining the identity of the allelic variant of a polymorphic regioncomprises contacting a nucleic acid of the subject with at least oneprobe or primer which is capable of hybridizing to an SR-BI gene.
 10. Amethod of claim 9, wherein the probe or primer is capable ofspecifically hybridizing to an allelic variant of the polymorphicregion.
 11. A method of claim 10, wherein the probe or primer is capableof specifically hybridizing to an allelic variant having a thymidine atposition 41 of exon 8 of the SR-BI gene.
 12. A method of claim 1,wherein the probe or primer has a nucleotide sequence from about 15 toabout 30 nucleotides.
 13. A method of claim 1, wherein the probe orprimer is a single stranded nucleic acid.
 14. A method of claim 1,wherein the probe or primer is labeled.
 15. A method of claim 1, whereindetermining the identity of the allelic variant of a polymorphic regionis carried out by allele specific hybridization.
 16. A method of claim1, wherein determining the identity of the allelic variant of apolymorphic region is carried out by primer specific extension.
 17. Amethod of claim 1, wherein determining the identity of the allelicvariant of a polymorphic region is carried out by an oligonucleotideligation assay.
 18. A method of claim 1, wherein determining theidentity of the allelic variant of a polymorphic region comprisesperforming a restriction enzyme site analysis.
 19. A method of claim 18,wherein the restriction enzyme is a HaeIII enzyme.
 20. A method of claim1, wherein determining the identity of the allelic variant of apolymorphic region is carried out by single-stranded conformationpolymorphism.
 21. A method for determining whether a female subject has,or is at risk of developing, an abnormally low HDL level, comprisingdetermining the identity of the allelic variant of a polymorphic regionof the SR-BI gene of the subject and comparing the allelic variant ofthe subject with allelic variants associated with low HDL levels, tothereby determine whether the subject has or is at risk of developing anabnormally low HDL level.
 22. A method of claim 21, comprisingdetermining the identity of the nucleotide at 41 in exon 8 and/ornucleotide 54 in intron 5, wherein the presence of a cytidine atposition 41 of exon 8 and/or the presence of a thymidine at position 54of intron 5 indicates that the subject has or is at risk of developingan abnormally low HDL level.
 23. A kit for determining whether a subjecthas, or is at risk of developing, a low HDL level, comprising a probe orprimer which is capable of hybridizing to an SR-BI gene and therebyidentifying whether the SR-BI gene contains an allelic variant of apolymorphic region which is associated with a low HDL level andinstructions for use in diagnosing a subject as having, or having apredisposition, towards developing a low HDL level.
 24. A kit of claim23, wherein the polymorphic region is located in an exon.
 25. A kit ofclaim 24, wherein the exon is exon
 8. 26. A kit of claim 25, wherein thepolymorphic region is a nucleotide polymorphism located at nucleotide 41of exon
 8. 27. A kit of claim 26, wherein the presence of a cytidine atnucleotide 41 of exon 8 of the SR-BI gene is indicative that the subjecthas or is at risk of developing an abnormally low HDL level.
 28. A kitof claim 23, wherein the polymorphic region is located in an intron, andwherein the subject is female.
 29. A kit of claim 28, wherein the intronis intron
 5. 30. A kit of claim 29, wherein the polymorphic region is anucleotide polymorphism located at nucleotide 54 of intron
 5. 31. A kitof claim 30, wherein the presence of a thymidine at nucleotide 54 ofintron 5 of the SR-BI gene is indicative that the subject has or is atrisk of developing an abnormally low HDL level.
 32. A kit fordetermining whether a female subject has, or is at risk of developing,an abnormally low HDL level, comprising a probe or primer which iscapable of hybridizing to an SR-BI gene and thereby identifying whetherthe SR-BI gene contains an allelic variant of a polymorphic region whichis associated with a low HDL level and instructions for use indiagnosing a subject as having, or having a predisposition, towardsdeveloping a low HDL level.
 33. A kit of claim 32, wherein thepolymorphic region is a nucleotide selected from the group consisting ofnucleotide 41 in exon 8 and nucleotide 54 in intron 5, wherein thepresence of, the presence of a cytidine at position 41 of exon 8 and/orthe presence of a thymidine at position 54 of intron 5 indicates thatthe subject has or is at risk of developing an abnormally low HDL level.34. A method for predicting the effect of hormone replacement therapy onthe HDL level in a female subject comprising: identifying one or moreallelic variants of the SR-B1 gene which are associated with abnormallylow HDL levels in females, thereby predicting the effect of hormonereplacement therapy on the HDL level in the subject.
 35. The method ofclaim 34, wherein hormone replacement therapy results in an abnormallylow HDL level.
 36. The method of claim 34, wherein the allelic variantscomprise a cytidine at position 41 of exon 8 and/or a thymidine atposition 54 of intron
 5. 37. The method of claim 34, wherein the femalesubject is postmenopausal.
 38. A method of predicting the effect ofhormone replacement therapy on a female subject, wherein theidentification of allelic variants of the SR-B1 gene which areassociated with abnormally low HDL levels in females results in aprediction that hormone replacement therapy will result in abnormallylow HDL levels.